Identification of glutamic acid 105 at the active site of Bacillus ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 35, Issue of December 15, pp. 25059-25066,1992 Printed in U.S.A.

Identification of Glutamic Acid 105 at the Active Site of Bacillus amyloliquefuciens 1,3- 1,4-&~-Glucan 4-Glucanohydrolase Using Epoxide-based Inhibitors*

(Received for publication, June 19, 1992)

Peter Bordier H#jjsg, Rosemary CondronS, John C. Traegerll, Joseph C. McAuliffeII , and Bruce A. Stone$ From the Departments of $Biochemistry and TChemistry, La Trobe University, Bundoora, Victoria 3083, Australia and the (ISchool of Chemistry, University of Western Australia, Nedlands 6009, Australia

Bacillus amyloliquefaciens 1,3-1,4-&~-glucan 4- glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [’*C](3,4)- epoxybutyl/3-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC) . The affinity label was esterified exclusively to the y-carboxylate of G1u1OS in the sequence Gly-Thr-Pro-Trp-Asp-mu-Ile-Asp-Ile- Glu”’. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in @-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of GluloS by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile’06 peptide bond. Iden- tification of pheny1thiohydantoin-Glulo6 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

The identification and location of catalytically active amino acid residues in enzymes is a challenge to biochemists and is often approached by using mechanism-based, active-site-directed inhibitors (1). One extensively used class of such inhibitors consists of a substrate analogue bearing an epoxide group that is susceptible to nucleophilic attack by catalytic amino acids. These inhibitors have been applied to kinases (2), proteases (3), and in particular to glycoside and polysaccharide hydrolases (4-20). More recently Z-deoxy-2-fluoro-~- glycopyranosides have also been applied to the study of glycosidases (21,22) (see Ref. 23 for a recent review on glycoside

* This work was supported by grants from the Australian Research Council (to P. B. H. and to B. A. S.) and by an equipment grant from La Trobe University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 To whom correspondence and reprint requests should be ad- dressed Dept. of Biochemistry, La Trobe University, Bundoora, Victoria, Australia 3083. Tel.: 61-3-479-2156; Fax: 61-3-479-2467.

hydrolase inhibitors). Because of their central role in biochemistry and carbohydrate technology polysaccharide hydrolases have been the subject of intensive studies and a large number of primary structures have been defined. For example, more than 50 genes encoding enzymes involved in xylan or cellulose hydrolysis have been cloned and sequenced (24). Although these studies have allowed extensive sequence comparisons, they have not provided significant information on the identity of catalytic amino acids. This is consistent with the absence of sequence similarities in catalytic amino acid- bearing peptides derived from the active site of many glycosidases (23) and glycosyltransferases (25). This lack of sequence homology in these functionally related hydrolases is perhaps not surprising since the three-dimensional structure of hen egg-white lysozyme revealed that the enzyme folds in such a way that most of the amino acid residues in sequential vicinity of the catalytic amino acids (Asp6’ and G ~ u ~ ~ ) do not interact with bound substrate (26). For these reasons homology searches are rarely productive in identifying catalytic amino acids and therefore we have taken another approach to this problem by employing a wide range of epoxyalkyl oligoglucosides for the irreversible active-site-directed inhibition of a series of P-D-glucan endohydrolases (10). These mechanism-based inhibitors act with a very high degree of specificity (10, 11) and form covalent linkages with nucleophilic catalytic groups (23). In this report we employ one such inhibitor to define a glutamate residue at the active site of a Bacillus amyloliquefaciens endohydrolase’ and describe a sensitive and non-radioactive strategy for the identification of active-site carboxylates in polysaccharide hydrolases susceptible to inactivation with epoxy-activated inhibitors.

EXPERIMENTAL PROCEDURES

Chemicals-Cellufine 2000 was purchased from Amicon Corp., Bio- Gel P-6 from Bio-Rad, Endoproteinase Glu-C from Boehringer and Promega, and trypsin from Worthington. Barley 1,3-1,4-P-D-glucan was obtained from Biocon, Australia.

Inhibitors-The mixture of diastereoisomers of peracetylated [“C] (R,S)-3,4-epoxybutyl 8-cellobioside (4.5 X 10’ cpm X nmol”) pre- pared from [“C]glucose (27) and (R,S)-3,4-epoxybutylP-cellobioside (28) were generous gifts from Drs. E. B. Rodriguez and R. V. Stick, University of Western Australia. Inhibitors were deacetylated im- mediately before use as described earlier (11).

Enzyme-The B. amyloliquefaciens endohydrolase was purified to homogeneity from a commercial source (Novo Ban SlOO) (29). The source of this enzyme is now known to be B. amyloliquefaciens (30)

The abbreviations used are: B. amyloliquefaciens endohydrolase, Bacillus amyloliquefaciens 1,3-1,4-@-D-glucan 4-glucanohydrolase; G4G-O-C4, (3,4)-epoxybutyl P-D-cellobioside; PTH, phenylthiohy- dantoin; MS, mass spectrometry; HPLC, high performance liquid chromatography.

25059

25060 Active-site Glutamate of B. amyloliquefaciens Endohydrolase

and not Bacillus subtilis as stated earlier (10, 11, 29, 31). The enzyme was assayed by determining the amount of reducing sugar equivalents released on incubation of the 8-glucanase with 0.5% (w/v) barley 1,3-

(10). 1,4-j%D-glucan at 40 'c in 50 mM sodium maleate buffer at pH 6.5

Preparation and Proteolytic Digestion of Inactivated B. amyloliquefaciens Endohydrohe-The B. amyloliquefaciens endohydrolase was inactivated at 37 "C by incubating the enzyme (13 mg/ml) in 50 mM sodium maleate (pH 6.5) containing [14C]G4G-0-C4 at a concentration of 25 mM. Inactivation using unlabeled G4G-0-C4 was performed under identical conditions except that the concentration of the enzyme was kept at 4 mg/ml. In both cases inactivation was stopped when approximately 10-20% activity remained. A homogeneous preparation of inactivated enzyme was then obtained by chromatography on a Cellufine 2000 column as described in the legend to Fig. 2. Inactivated enzyme eluted from this column was concentrated by centrifugal ultrafiltration in an Amicon CD 10 unit and, in the case of preparations inactivated with labeled inhibitor, further purified by chromatography on a Bio-Gel P6 column (1.5 X 12 cm) equilibrated in 100 mM sodium acetate buffer at pH 5.0. Prior to proteolytic digestion the B. amyloliquefaciens endohydrolase was denatured by precipitation with ice-cold trichloroacetic acid at a final concentration of 10% (w/v). After a 15-min incubation on ice the precipitated protein was collected by centrifugation for 15 min at 13,000 X g. The supernatant was discarded and the resulting pellet washed twice with ice-cold ethano1:ethyl acetate (2:1, v/v). More than 90% of the covalently linked inhibitor present prior to precipitation was recovered in the pellet. The protein pellets were dissolved in 100 mM NH4HC03, 0.05% SDS (w/v) at a concentration of about 4 mg/ml. Digestion of the protein in this buffer with either trypsin or endoproteinase Glu- C at a protease to substrate ratio of about 1:15 was performed at 37 "C.

High Performance Liquid Chromatography-Peptides from proteolytic digests were purified by reversed-phase HPLC employing a Brownlee RP-300 AQUAPORE C8 guard column (4.6 X 30 mm) connected with either a Phenomenex W-POREX 5 C8 (4.6 X 250 mm) or a (4.6 X 250 mm) 5-pm BakerbondTM WP C8 column (J. T. Baker Research Products). All elutions were at 1 ml/min with linear gradients from 0.1% aqueous trifluoroacetic acid up to 80% aqueous acetonitrile in 0.1% trifluoroacetic acid.

Electrosprayzonization Mass Spectrometry-Samples (5-200 pmol) in 10 pl of MeOH:HzO:acetic acid (49.5:49.5:1, v/v/v) or in 10 pl of CH3CN:Hz0:acetic acid (49.5:49.5:1, v/v/v) were introduced at a flow rate of 2 pl/min into a VG BIO-Q electrospray mass spectrometer (VG-Biotech, Manchester, United Kingdom). The quadrupole mass spectrometer was scanned between 15 and 45 times (10 s/scan) to obtain the final spectrum, and the quadrupole was set for unit mass resolution. The mass scale was calibrated using the multiple charged ions from a separate introduction of horse heart myoglobin (Sigma). Following electrospray mass spectrometry the average molecular mass was calculated from the equation P,z, = M, + 1.0079 zi (32). All molecular masses are reported as average rather than monoisotopic masses.

Additional Analytical Techniques-Isoelectric focusing and amino acid analysis were performed as described (33) and amino acid sequencing by sequential Edman degradation was conducted in an Applied Biosystems 470.4 gas phase sequenator. PTH-derivative analysis was performed on a Zorbax C8 column at 61 "C (34). Base hydrolysis of inhibitor-peptide conjugates was performed at 28 "C for 6 h following addition of 0.1 volume of 1 M NaOH to appropriate HPLC fractions. All other analytical techniques were as described previously (10).

RESULTS

Purification and Characteristics of the B. amyloliquefaciens Endohydrohe-The B. amyloliquefaciens endohydrolase was purified from a crude commercial enzyme preparation (Novo Ban S100) derived from the culture medium of B. amyloliquefaciens (30). Analysis of the preparation by SDS-polyacryl- amide gel electrophoresis revealed the presence of a single 25- kDa component (data not shown). Amino-terminal sequencing of the protein preparation revealed a single sequence:

NHZ-QTGGSFFEPF" SEQUENCE 1

This sequence corresponds exactly to that deduced from the nucleotide sequence of the B. amyloliquefaciens gene encoding pre-1,3-1,4-P-D-glucan 4-glucanohydrolase and also es- tablishes the site of presequence cleavage suggested earlier (35). However, analysis of the purified preparation by electrospray mass spectrometry revealed two distinguishable protein species: a major component (about 80%) of M, 24,149.2 & 1.1 S.D. and a minor (about 20%) of M, 24,305.7 k 3.9 S.D., respectively (Fig. 1, A and B) . The calculated molecular mass based on the published gene structure (35), the amino-terminal sequence, and the average mass of individual amino acids of the enzyme is 24,305.9 Da, which is in excellent agreement with that determined for the minor component by mass spectrometry. The difference in molecular mass between the two species is 156.5 f 5.0 Da, corresponding to the molecular mass of an arginine residue (average molecular mass 156.19 Da). Support for this contention was provided by analysis of the enzyme preparation by isoelectric focusing, which revealed the presence of a major species of PI 8.1 and a minor species of PI 8.3 (data not shown). Furthermore, chromatography of the preparation by chromatofocusing led to the separation of two components eluting at pH 8.0 and pH 7.9, respectively. The acidic component was the more abundant, but both proteins were highly active (data not shown). Extensive sequencing of peptides derived from the mixture of the two components was in excellent agreement with the published gene sequence for the B. amyloliquefaciens endohydrolase as was amino acid analysis, except that the arginine content was lower than predicted (data not shown). We conclude that the two components represent isoforms of the same enzyme pos- sibly differing by only one arginine residue. Whether these two isoforms are derived from a single or two highly related B. amyloliquefaciens strains is not clear. However, the two forms are practically identical and additional work described in this paper employed the preparation containing both the

I I . I 800 1000 1200 1400 1600 1800 24OOO 24500 25000 25500

I ' I I ' I

I 1 L . I

mlz mass

FIG. 1. Electrospray ionization mass spectrometry of B. arnyloliquefaciens endohydrolase. A, spectrum of the native B. amyloliquefaciens endohydrolase, which contained two major species (land ZZ) of M, = 24,149.2 5 1.1 and M, = 24,305.7 5 3.9, respectively. The M , values were calculated using eight sets of (z,, Pi) values for component I and eight sets of (zL, Pi) values for component 11. B, a computerized transform of the data shown in panel A onto a real mass scale. C, spectrum of the inactivated B. amyloliquefaciens endohydrolase, which contained one major species (I) of M, = 24,561.3 k 1.5 and two minor components of M, = 24,713.8 k 8.5 and M, = 24,974.8 5 3.6, respectively. The M, values were calculated using six sets of (z,, Pi) values for component I, nine sets for component 11, and seven sets for component 111. D, a computerized transform of the data shown in panel C onto a real mass scale.

loo0 I200 1400 1600 24oM) 24500 25000 25500

Active-site Glutamate of B. amyloliquefaciens Endohydrolase 25061

M , 24,149 and M, 24,306 components. Similarly, since the primary structure of B. subti1i.s 1,3-1,4-@-D-glucan 4-glucanohydrolase shows 95% positional identity to that of the B. amyloliquefaciens endohydrolase over the entire length of the molecules (35, 36), the conclusions drawn from the work reported here and in previous publications (10, 11) will apply equally to the enzymes recovered from either source.

Preparation and Isolation of Inactivated B. amyloliquefaciens Endohydrolase-Our previous studies of the inhibition of the B. amyloliquefaciens endohydrolase (10, 11) demonstrated that the epoxybutyl-@-cellobioside, designated G4G- O-C4, was a specific and mechanism-based, active-site-directed inhibitor of this enzyme. For the identification of active-site residues the enzyme was therefore incubated for a 4-h period with [l4C]G4G-O-C4 to minimize the risk of form- ing nonspecific inhibitor-enzyme adducts and approximately 80% inactivation was obtained (data not shown). The remaining active enzyme was removed by application of the partially inactivated enzyme preparation to a gel filtration column packed with a cellulosic matrix (Fig. 2). The native B. amyloliquefaciens endohydrolase interacts strongly with cellulosic matrices (29); its elution volume was at least twice that of the column bed volume (V,) and significantly greater than that of cytochrome c, despite its much higher molecular mass (24 versus 12 kDa, data not shown). The inactivated endohydrolase exhibits a lower degree of affinity for the cellulosic matrix and has an elution volume equal to Vt (Fig. 2).

Stoichiometry of Inhibitor Binding-After isolation by gel filtration chromatography the B. amyloliquefaciens endohydrolase was chromatographed on a Bio-Gel P6 column to remove any remaining unincorporated, labeled inhibitor (not shown). The radioactively labeled protein was recovered and concentrated to a final volume of 650 pl by centrifugation in a n Amicon CDlO unit, without loss of radioactivity (9.8 X lo4 cpm). The amount of inhibitor and protein in the concentrated preparation was determined by liquid scintillation counting and quantitative amino acid analysis, respectively,

n CONrFX

li

2 6 10 14

FRACTION NUMBER

FIG. 2. Separation of native and inactivated B. amyloliquefaciens endohydrolase. CONTROL, B. amyloliquefaciens endohydrolase (800 pg, 1 mg/ml) in 50 mM sodium maleate a t pH 6.5 was applied to a 1 X 9-cm column of Cellufine 2000 equilibrated in and eluted with 100 mM NaAc at pH 5.0. The column was operated at room temperature a t a flow rate of 7.5 ml/h and continuously moni- tored at 280 nm. Fractions (0.75 ml) were collected and assayed for activity, and polypeptide content by sodium dodecyl sulfate polyacryl- amide gel electrophoresis. INHIBITED, same as for the control panel except that 800 pg of endohydrolase previously inactivated to about 20% residual activity with GIG-0-C, was applied to and chromatographed on the Cellufine column. Sodium maleate elutes in peak A , inactivated enzyme in peak B, and native enzyme in peak C.

and 219 nmol of inhibitor and 195 nmol of endohydrolase were recovered. The inactivated enzyme preparation was further characterized by electrospray mass spectrometry (Fig. 1, C and D ) . As described earlier, analysis of control enzyme gave an estimated M, of 24,149.2 f 1.1 S.D. with a minor component of M, 24,305.7 f 3.9 S.D., whereas that of the inactivated preparation revealed a major component (80%) of M, 24,561.3 f 1.5 S.D. and two less prominent components of M, 24,713.8 f 8.5 S.D. and 24,974.8 f 3.6 S.D., respectively. The difference in molecular mass between the major component of the inactivated and control preparations, respectively, is 412.1 f 2.6 Da, and that between the 24,713.8 and the 24,305.7 components of the control and inactivated preparation, respectively, is 408.1 f 12.4 Da. These differences can be compared to the 412.1 Da for the inhibitor itself. The M, 24,974.8 component presumably corresponds to a minor part of the 24,149.2 component, which carries two molecules of covalently linked inhibitor, as indicated by the difference of 825.6 f 4.7 Da (Fig. 1). Based on the stoichiometry of labeling and the mass spectral data it may be concluded that essen- tially all enzyme molecules of the inactivated preparation carry only one covalently bound inhibitor molecule. The presence of the M, 24,974.8 component, however, indicates that prolonged incubation may lead to modification with at least two bound inhibitor molecules per enzyme moiety and ac- counts for the slightly high ratio of inhibitor to protein (1.l:l.O).

GIG-0-C4 Is Linked in an Alkali-sensitive Linkage to Glu'05 of B. amyloliquefaciens Endohydrolase-Preliminary experiments showed that the B. amyloliquefaciens endohydrolase was exceedingly resistant to protease degradation, and we found that satisfactory proteolysis was obtained only following trichloroacetic acid precipitation and subsequent incubation with proteases in SDS-containing buffers. In the final procedure, inactivated enzyme recovered from the Bio-Gel P6 column was precipitated at 0 "C with 10% (w/v) trichloroacetic acid and digested for 4 h at 37 "C with endoproteinase Glu-C. The resultingpeptides were resolved by reversed-phase HPLC and the radioactivity of the fractions determined by liquid scintillation counting (Fig. 3). In three separate experiments radioactive material was detected in only two fractions (V1 and V2). The total amount of radioactivity recovered in these fractions was 70% of that present prior to proteolysis. The radioactive label was distributed equally between the two fractions, which each contained a single component. Amino acid sequence analysis of the material in peak V1 revealed the following sequence.

GTP(W)DXIDIE'"

SEQUENCE 2

This sequence corresponds exactly to that of residues 100- 109 for the mature enzyme predicted from the nucleotide sequence of the endohydrolase except that the glutamic acid residue at position 105 was not detected in the peptide sequence. The most likely interpretation of this result is that the G4G-0-C4 inhibitor is bound in ester linkage to Glulo5 of the inactivated enzyme. This linkage is stable in the conditions employed during automated sequencing but the resulting PTH-derivative released is not identifiable and cleavage with endoproteinase Glu-C does not take place at the modified Glulo5 residue.

The predicted M, of the peptide-inhibitor adduct (shown in Scheme 1) is 1574.1 and that determined by electrospray mass spectrometry of this peptide fragment was 1574.4 (data not shown).


T I 75 T2

YEVRMKPAKNTGIVSSFFTYTGPTEGTPWDEIDIEFLGKDTTK 1 v2 V I

0.4 OD214

1 A, I 30'

I 60'

I 90'

FIG. 3. Reversed-phase HPLC elution profile of endoproteinase Glu-C digests of ['4C]G4G-O-Cs-labeled B. amylolique- faciene endohydrolase. ["C]G4G-O-C4-inactivated B. umylolique- fuciens endohydrolase (475 pg) partially digested for 4 h with endoproteinase Glu-C was mixed with an equal volume of 6 M guanidinium chloride and analyzed by chromatographyon a Phenomenex W-Porex 5 Cs column. After sample injection the column was washed for 5 min in solvent A (0.1% trifluoroacetic acid, v/v) and then eluted with a linearly increasing gradient of solvent B (0.1% (v/v) trifluoroacetic acid, 9.9% (v/v) HzO, 90% (v/v) CH3CN). The gradient was composed of the following linear segments: 0-60% B from 5 to 120 min, 60% B from 120 to 130 min, and 60-90% B from 130-140 min. Fractions were collected and their radioactive content estimated by scintillation counting. Radioactivity (70% of the loaded material) was found to be associated with only two peptide peaks, V1 and V2. Ten cycles of automated Edman degradation of the material recovered in peak V1 and V2 gave the single unique sequences shown. The position of peptides V1 and V2 and T1 and T2 (discussed under "Results") within the primary structure of the B. amyloliquefaciens endohydrolase is shown for convenience.

NHZ-GTPWDEIDIE-COOH I

O-Cd-OG4G SCHEME 1

The binding of the inhibitor in an ester linkage is further supported by our observations that a significant increase in the PI of the B. amyloliquefaciens endohydrolase accompanies inactivation (data not shown). Such a difference in PI between active and inactive enzyme can be exploited to separate inactive and active enzyme by chromatofocusing or ion ex- change chromatography in cases where suitable affinity matrices are not available.

Incubation of the isolated peptide (Vl) with NaOH generated a component which eluted as a single non-radioactive peak during reversed-phase HPLC, but at a slightly higher acetonitrile concentration than the parent compound (data not shown). Amino acid sequence analysis of this material yielded the following sequence.

GTP(W)DEIDIE'W SEQUENCE 3

This sequence is identical to that obtained prior to NaOH

treatment except that Glu'05 now was clearly identifiable at position 6. The predicted M, for this compound is 1174, in complete agreement with that of 1174 obtained by electrospray MS (data not shown). It is concluded that G4G-O-C4 binds to GIulo5 during enzyme inactivation.

The material in peak V2 (Fig. 3) was recovered and the sequence of the first 9 residues determined.

VRMKPAKNTGS6 SEQUENCE 4

This corresponds to a peptide generated by cleavage at G ~ u ~ ~ . Since the V2 peptide carried 50% of the associated label it was expected to represent a partial digest spanning residues 77-109 (see Fig. 3). Amino acid analysis of this peptide was in exact agreement with this proposition, and the predicted M, of 4,101.6 for the peptide carrying the inhibitor was in agreement with the 4,101.6 determined by electrospray MS (data not shown).

Although it is unlikely, it is formally possible that the peptide recovered in peak V2 carried the inhibitor esterified to Glug9, a residue that is not conserved in any of the homologous endohydrolases from other species and therefore not expected to be catalytically active (Fig. 5). To address this question we also digested the labeled protein with trypsin and recovered two labeled peaks (T1 and T2), which accounted for at least 70% of the label associated with the protein prior to digestion (data not shown). The amount of label associated with peak T2 was twice that recovered in peak T1. Sequence analysis of the first 27 residues in T2 revealed a single sequence starting at Amw.

&LNTGIVSSFFTYTGPTEGTP(W)DXIDIEF'lo SEQUENCE 5

This sequence includes that of peptide V1. The assignment of PTH-Glu" and PTH-Glu"' in this peptide was unambig- uous, whereas PTH-Glulo5 (data not shown) was barely de- tectable. This shows that the only residue affected by the inhibitor is Glulo5, even in peptide V2, which spans residues 77-109. Amino acid analysis confirmed that peptide T2 cor- responded exactly to a peptide spanning residues 85-113 of the mature protein (data not shown). Sequence and amino acid analysis of the labeled material in T1 showed that it represented a partial tryptic digestion product which, like T2, started at Asn= but extended beyond Lys1l3 to Lys117 (data not shown). The locations of peptides V1, V2, T1, and T2 in the primary structure of B. amyloliquefaciens endohydrolase are summarized in Fig. 3.

Comparative Peptide Mapping of Active and Inactivated Enzyme Can Reveal the Point of Inhibitor Attachment-The results obtained during the analysis of ['4C]G4G-O-C4-labeled enzyme show that endoproteinase Glu-C-mediated peptide hydrolysis is blocked at glutamate residues modified with the inhibitor. Thus, a comparison of endoproteinase Glu-C-generated digestion patterns obtained from control and inactivated proteins, respectively, could allow the identification of peptides that arise solely as a consequence of modification by G4G-0-C4 of the protein. A criterion for selection of a true active-site-derived peptide would be that sequencing of the peptide revealed the lack of an identifiable glutamate residue prior to but not following base treatment. To test the feasi- bility of this approach the B. amyloliquefaciens endohydrolase was inactivated with non-radioactive G4G-OX4 and chromatographed on the Cellufine column to obtain a homogeneous, inactivated preparation. Following trichloroacetic acid precipitation, inactivated and control enzyme were digested for 17 h at 37 "C at a protease to substrate ratio of 1:20. The


resulting peptide mixtures were analyzed by reversed-phase HPLC (Fig. 4). The peptide elution profiles obtained from the two preparations were identical except for two components. One component (labeled * in Fig. 4A) was present almost exclusively in the control digest, and another component (labeled * in Fig. 4C) was present only in the digest of the inactivated enzyme preparation. The assignment of these components as specific to the respective digests was confirmed by mixing experiments employing 50% of each digest mixture and chromatography under identical conditions (Fig. 4B). As expected, the relative intensity of most peaks remained un- changed as a result of mixing, but the intensity of the two specific components had halved. The peptide peak specific to the digest of the inactivated preparation was recovered and sequenced and shown to give the sequence GTP(W)DXIDIE- COOH, which is identical to that of peak V1 recovered from the radioactive inhibitor experiments. No peak related to that of the partially digested V2 peptide (seen in Fig. 3) was found in these samples subjected to more extensive digestion. Se- quence analysis of the component found predominantly in the control digest gave the sequence GTP(W)DE-COOH, showing

I C I I I

FIG. 4. Comparative peptide mapping of control and affinity-labeled B. amyloliquefaciens endohydrolase. Control (panel A ) or G4G-O-C4-inactivated (panel C) B. amyloliquefaciens endohydrolase at a concentration of 2 mg/ml was digested for 17 h with endoproteinase Glu-C and 20 p1 (i.e. 40 pg) of the resulting digest analyzed by reversed-phase HPLC on a BakerbondTM C8 column preceded by a C, guard column. A mixture of the two digests (10 pl each) was also analyzed under identical conditions (panel B ) . The elution conditions were identical to those described for Fig. 3. Peptide peaks unique to the digest of the control (*) and inactivated preparation (*) were recovered and fully sequenced.

B:

F. succinogenes ADGRPWVEVDf55 i 4 4 j B . circulans glcA c. thermocellum celc HFDTFITEKD?. (61'

GTWAASGEIDV555 ( 6 0 )

Bacillus sp. strain 1139 DGGPYFDERDVZY4 Cryptococcus albidus GLEVPMTELDV217 ( 6 3 )

(62)

Clostridium thermocellum GVIVSFTEID1757 ( 6 4 ) C. fimi GVDVRITELD1277 (21) Agrobacterium sp. PECYITENGA362 (22)

\ ",

FIG. 5. Alignment of B. amyloliquefaciens active-site sequence with sequences of microbial 8-glycanases. A, relevant portions of the deduced primary structures of the 1,3-1,4-fi-D-glUCan endohydrolases from F. succinogenes (44), B. macerans (45), B. amyloliquefaciens (35), B. subtilis (36), B. licheniformis (58), C. thermocellum (59), and T4 lysozyme (53) were aligned and identical residues shaded. The position of the affinity-tagged Glulo5 is indicated (*). B, conserved sequences in B. amyloliquefaciens (35) and F. succinogenes (44) 1,3-1,4-fi-D-ghcan endohydrolases, in a B. circulans (60) 1,3-@- D-glucan endohydrolase, in C. thermocellum (61) and Bacillus sp. strain 1139 (62) cellulases, in C. albidus (63) and C. thermocellum (64) xylanases, in a C. f imi exo-l,4-cellulase (22), and in an Agrobac- terium sp. fi-glucosidase (23) were located by inspection of published sequences. The underlined glutamate residues have been identified as likely catalytic nucleophiles using mechanism-based active-site directed inhibitors (this work and Refs. 22 and 23), and the residues occurring with a frequency greater than or equal to 45% in a given position are shaded. In this analysis the Ile, Leu, and Val residues are considered equivalent. The numbers to the right of each peptide refer to the position of the last residue of each peptide in the overall sequence of the respective enzymes.

that endoproteinase Glu-C cleavage does take place at the G 1 ~ ' ~ ~ - I l e ' ~ ~ peptide bond in the absence of G4G-0-C4 modification. These results are in complete agreement with those obtained above and show that the identification of active-site residues in polysaccharide hydrolases can be achieved with non-radioactive inhibitors. In separate studies we have successfully extended this approach to identify active-site glutamate residues in several plant @-glucan hydrolases without the use of radioactive inhibitow2

The GIU''~ Nucleophile Forms Part of a Consensus Sequence Conserved in a Large Number of @-Glucanases-Recently Tu11 et al. (21) used 2',4'-dinitrophenyl 2-deoxy-2-fluoro-@-~-glu- copyranoside as an affinity label to identify G ~ u ~ ~ ~ as the nucleophile in the active site of a Cellulomonas fimi @-1,4- exoglucanase. The nucleophilic glutamate is conserved in the consensus sequence Glu-Leu/Ile-Asp-Ile in 10 members of the glycanase F family (21). It is also conserved in the sequence around Glu'05 of the B. amyloliquefaciens endohydrolase ex- amined here and to a lesser extent in a sequence around the catalytic G ~ u ~ ~ ~ of an Agrobacterium sp. glucosidase (22) (Fig. 5 ) . Inspection of a number of other prokaryote endoglucanase sequences has revealed the widespread, but not universal, occurrence of this consensus sequence (Fig. 5 ) . Presumably, these sequences are functionally related to the catalytic sites of the enzymes.

DISCUSSION

Although a catalytic role for carboxyl groups has been firmly established in a number of glycosidases, glycosyl trans-

' L. Chen, G. B. Fincher, abd P. B. Hey, unpublished observations.


ferases, and polysaccharide hydrolases, the identification of specific catalytic carboxylates within these enzymes has only been achieved in a restricted number of examples, none of which include the P-glucan endohydrolases (see Refs. 21, 25, 23, and 37 for a review). Sequence comparisons may now be used to locate consensus sequences of the type identified in the C. fimi, B. amyloliquefaciens, and Agrobacterium sp. enzymes using mechanism-based affinity reagents (Fig. 5). How- ever, the absence of this sequence in the majority of polysaccharide hydrolases precludes the general use of this approach. For example, while the B. amyloliquefaciens and barley 1,3- 1,4-@-D-glucan 4-glucanohydrolases exhibit identical substrate specificities and action patterns (31, 37, 38), they share no regions of sequence similarity (35,36, 39) and their differ- ential response to optically pure inhibitors imply that they may employ sterically distinct mechanisms to achieve glycoside bond hydrolysis (11). Similarly, cellulases readily hydro- lyze 1,3-1,4-@-D-glucans, but no obvious sequence similarities exist between cellulases and 1,3-1,4-@-D-glucanases derived from the same species (see e.g. Refs. 40 and 41). A glutamate residue in a conserved Asn-Glu-Pro sequence of two highly divergent endo-P-1,4-glucanases from B. polymoxa and B. subtilis is claimed to be important for enzymatic activity (42), but homologous sequences cannot be found in B. subtilis and B. amyloliquefaciens 1,3-1,4-P-D-glucan 4-glucanohydrolases.

The elucidation of three-dimensional structures of proteins at atomic resolution is another potential avenue for the identification of catalytic amino acids. Although catalytic residues at the active site of hen egg-white lysozyme were correctly identified from the protein structure (26), this approach has not always been applied successfully, especially in larger and more complex glycoside hydrolases such as cellobiohydrolase I1 from Trichoderma resii (43). These observations therefore suggest that the development of a general approach which combines the use of mechanism-based active-site-directed inhibitors followed by site-directed mutagenesis is necessary to define the catalytic components of polysaccharide hydrolases in particular, and enzymes in general.

Recently, we have established that @-glucan endohydrolases with distinct but related substrate specificities can be inactivated by mechanism-based epoxyalkyl &glycosides with a very high degree of specificity (10). For example it was found that (3s)-epoxybutyl cellobioside inactivates the B. amyloliquefaciens endohydrolase less efficiently than does the (3R)- isomer, whereas the reverse is true for the barley 1,3-1,4-/3- glucan 4-glucanohydrolase isoenzyme I1 (11). The high degree of inhibitor specificity and the wide range of potential target enzymes will ensure the general usefulness of this class of inhibitors in the identification of active-site residues.

Here, the inhibitor [14C]G4G-0-C4 was employed to identify Glu'" in the sequence GTPWDEIDIE'OS at the active site of the B. amyloliquefaciens endohydrolase. Glu105 is the only residue quantitatively modified by the mechanism-based inhibitor, and, once modified, the enzyme could no longer hy- drolyze 1,3-1,4-P-D-glUCan. Binding of the inhibitor was ac- companied by a reduction but not elimination of the enzyme's affinity for a cellulosic polymer. This shows that inhibitor action is aimed at the substrate binding site of the enzyme, but the residual binding to the cellulosic matrix indicates that an extended substrate binding cleft exists as the employed inhibitor covers only three glucosyl binding subsites (10). The complete loss of activity upon esterification of Glulo5 is most likely due to the loss of the enzyme's catalytic nucleophile (23), but it could in principle also be due to a blockage of the substrate binding site by the glucosyl-residues of the inhibitor. Site-directed mutagenesis of the B. amyloliquefaciens endohy-

drolase could establish the degree to which enzyme catalysis is dependent on Glulo5.

Extended sequence homology between the 1,3-1,4-@-D-glU- can 4-glucano-hydrolases from Bacillus spp. and related enzymes from other sources has been noted in the case of a 1,3- 1,4-P-D-glucan 4-glucanohydrolase from Fibrobacter succinogenes (44). A limited similarity comprising the catalytic region of T4 lysozyme has also been suggested (Ref. 45, Fig. 5). The sequence similarity between the F. succinogenes and Bacillus enzymes is about 40% and extends throughout the two enzymes (44). The two types of enzyme have identical substrate specificities but differ somewhat in their pH optima. The pH optimum of the F. succinogenes enzyme is broad with a maximum between 5.75 and 6.25 (46) as compared to a more well defined optimum at 6.5 for the Bacillus enzyme (47). The relatively high pH optimum for the B. amyloliquefaciens endohydrolase compared with most other carbohydrases (see Ref. 37) could be explained by a comparatively high value for the pK, of the postulated catalytic nucleophile (Glu105). The absence of basic amino acids and the abundance of negatively charged amino acids in the vicinity of Glulo5, in particular Asplo4, will result in a significant increase in the pK, of Glulo5 and thus a higher pH optimum. It is thus predicted that the introduction of a basic amino acid in the vicinity of Glulo5 and/or elimination of Asplo4 would lower the pK, value of Glulo5 and thus broaden the pH optimum of the B. amyloliquefaciens endohydrolase to enhance the activity at, e.g., pH 5.0. Two lines of evidence suggest that removal of Asplo4 indeed could result in the creation of an endohydrolase with enhanced activity at pH 5. First, the F. succinogenes enzyme contains a conserved glutamate (Glu") corresponding to Glulo5 of the Bacillus enzymes, but a valine residue in the position corresponding to Asp'04 and an Arg residue in the position corresponding to Thr"' (Fig. 5). Thus the pK, of F. succinogenes endohydrolase Glu5' is expected to be lower than that of Glulo5 in the B. amyloliquefaciens enzyme, which in turn would result in both a lower and a broader pH optimum for the former enzyme, as observed. Second, Sierks et al. (48) showed that substitution of the noncatalytic G1ulm with a Gln residue in the active site of Aspergillus awamori glucoamylase led to a reduction of the pH optimum from 4.5 to 3 most likely due to a lowering of the pK, of the neighboring Glu17$ residue expected to serve as a catalytic proton donor.

The identity of the proton donor in the B. amyloliquefaciens endohydrolase is not known. It is to be expected that the pK, of the proton donor is significantly higher than that of Glulo5 (the likely catalytic nucleophile) and may therefore not be represented by an aspartic acid residue but rather by another glutamic acid residue or even a protonated histidine residue. A histidine has been implicated at the active site of an 1,3-@- D-glUCan 3-glucohydrolase from Basidiomycete sp. QM806 (49) and an endoglucanase D from Clostridium thermocellum (50). Sequence comparisons of the B. amyloliquefaciens endohydrolase with T4 lysozyme (45) revealed a similarity in a region containing the postulated catalytic nucleophile (Asp2') and catalytic acid (Glu") of the latter (51). It is doubtful, however, whether this sequence alignment has any functional relevance, inasmuch as the likely catalytic nucleophile of the Bacillus enzymes identified in this paper was identified as the putative catalytic acid by Borriss et al. (45) based on the alignment with the sequence of the lysozyme T4 (Fig. 5). Furthermore, even though the catalytic nucleophile of the lysozyme molecule matched that of Asplo4 in the Bacillus enzymes, an asparagine residue is found in the corresponding position of the F. succinogenes enzyme (Fig. 5). The recent crystallization (52) of the B. amyloliquefaciens endohydrolase


and our identification of the likely catalytic nucleophile may lead to the identification of the catalytic acid, which is expected to be in close proximity to Glulo5 as has been shown for lysozyme (26) and suggested for Taka-amylase (53).

For the unequivocal assignment of catalytically important amino acids following inactivation with mechanism-based inhibitors, it is essential that the stoichiometry of inhibitor binding is known, that the inhibitor-enzyme bond is stable, and that the inhibitor can be detected easily (23). For these reasons it is in most cases only practical to perform the experiments employing radioactive inhibitors. The synthesis of radioactive inhibitors is often a major undertaking, and in some cases the specific radioactivity of the final product is relatively low. For example, the specific activity of inhibitors synthesized for the identification of Glulo5 in this report, in hen egg white lysozyme (7), and Glu'% in soybean /3- amylase (18) were 450, 110, and 48 cpm/nmol, respectively. Detection of labeled peptides and unequivocal identification of modified amino acids therefore requires a large amount of purified protein and precludes the investigation of a significant number of enzymes. As an alternative to radioactive inhibitors, we found here that the carboxylate modified by non-radioactive inhibitors may be identified quite easily. The approach takes advantage of the high stability of the covalent inhibitor-enzyme linkage and the resistance toward endoproteinase Glu-C mediated cleavage of peptide bonds involving a glutamate residue modified by the inhibitor. HPLC mapping of proteolytic digests derived from inactivated and control samples quickly reveals the peptide susceptible to inhibitor modification. Subsequent sequencing of the modified peptide shows that no recognizable PTH-derivative can be detected a t positions corresponding to the modified glutamate, indi- cating resistance of the ester linkage to breakdown during the many cycles of Edman degradation (>20) in the gas-phase sequenator. This stability was also noted by Nitta et al. (18) and is in marked contrast to the lability of the adducts obtained with the conduritol-B and -C epoxides (9, 54, 55) . Further confirmation of the point of inhibitor attachment is obtained by base hydrolysis to confirm the presence of the modified glutamate in the peptide backbone. The sequencing of peptides unique to the digest of control enzyme confirms the cleavability of the peptide bond involving the a-carboxylic acid at the catalytic Glu residue prior to modification. If the polysaccharide hydrolase investigated is modified at a catalytic aspartate the methodology should prove equally appli- cable. Endoproteinase Asp-N would be used instead of endoproteinase Glu-C. Although the use of a protease which rec- ognizes residues involving the modified amino acid are likely to provide the most clearcut differences in HPLC peptide maps between inhibited and control enzyme, other proteases, such as trypsin, can also be used. We and others (56) have found that the attachment of a sugar-containing inhibitor to a peptide will generally lower the retention time of that peptide on reversed-phase columns. Because of the very high sensitivity of modern HPLC and sequencing technology (57), the use of comparative peptide mapping will require much less starting material than does the use of radioactive inhibitors of low specific activity. As demonstrated in the present paper, electrospray mass spectrometry can be employed to assess the stoichiometry of inhibitor binding at high sensitivity. Together the techniques outlined in this work may be applied to any enzyme for which specific covalently bound inhibitors are available and should therefore facilitate the definition of essential amino acids in enzymes of interest.

Acknowledgments-We are indebted to Drs. R. V. Stick and E. B. Rodriguez for the generous gift of inhibitors used in this work. We

thank Dr. G. B. Fincher for stimulating discussions.

Addendum-Following the completion of this manuscript, Planas et ul. (65) communicated that substitution of G1ul= with a Gln residue in the highly homologous Bacillus licheniformis 1,3-1,4-fl-D-glUCan 4- glucanohydrolase (see Fig. 5 ) completely abolished enzyme activity, while substitution of Asp133 with an Asn residue had limited effect on substrate binding and catalysis. These results are consistent with our assignment of Glu105 as the likely catalytic nucleophile in the B. amyloliquefaciens endohydrolase.

REFERENCES

2. Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 264,1879-1886 1. Plapp, B. V. (1982) Methods Enzymol. 87,469-499

3. Tsuru, D., Shimada, S., Maruta, S., Yoshimoto, T., Odo, K., Murao, S., and

4. Bause, E., and Legler, G. (1980) Biochim. Biophys. Acta 626,459-465 5. Clarke, A. J. (1988) Biochem. Cell €501. 6 6 , 871-879 6. Datema, R., Romero, P. A., Legler, G., and Schwarz, R. T. (1982) Proc.

Natl. Acad. Sci. U. S. A. 79,6787-6791 7. Eshdat, Y., McKelvy, J. F., and Sharon, N. (1973) J. Biol. Chern. 2 4 8 ,

5892-5898 8. Grabowski, G. A,, Osiecki-Newman, K., Dinur, T., Fabbro, D., Legler, G.,

Gatt, S., and Desnick, R. J. (1986) J. Biol. Chern. 261,8263-8269 9. Herrchen, M., and Legler, G. (1984) Eur. J . Biochem. 138,527-531

10. Hei. P. B.. Rodrieuez. E. B.. Stick. R. V.. and Stone. B. A. (1989) J. Biol.

Iwanaga, S. (1986) J. Biochern. (Tokyo) 99,1537-1539

.&ern. 264,4939-4947 '

J. Biol. Chem. 266 , 11628-11631 11. Hcij, P. B., Rodriguez, E. B., Iser, J. R., Stick, R. V., and Stone, B. A.

12. Isoda, Y., and Nitta, Y. (1986) J. Biochem. (Tokyo) 99,1631-1637 13. Is@?, Y.A.-,As,B,n,a, S., Takeo, K., and Nitta, Y. (1987) Agric. Biol.

14. Isoda, Y., and Nitta, Y. (1988) Agric. Biol. Chem. 62,271-272 15. Leeler. G. (1968) HoDw-Sevkr's 2. Phvsiol. Chern. 349.767-774

01, JZZd-dZZY

16. Legler; G., and Bau, E. (f973) Carbohydr. Res. 28,45152 17. Newburg, D. ,S., Yatziv, S., McCluer, R. H., and Raghavan, S.

18. Nitta, Y., Isora, Y., Toda, H., and Sakiyama, F. (1989) J. Biochem. ( Biochm. Bwphys. Acta 877,121-126

105.573-576

(1991)

Chem.

(1986)

Tokyo)

19. Thomas, E. W., Mckelvy, J. F., and Sharon, N. (1969) Nature 222 , 485-

20. White. W. J.. Jr.. Schrav. K. J.. Leeler. G.. and Alhadeff. J. A. (1986) 486

~~ ~~~~

andAbbersold,Rl ( 22. Withers, S. G., Warrt

and Aebersc 23. Legler, G. (1990)Adu. Carbohydr. Chem. 48,319-384 24. Beguin, P. (1990) Annu. Reu. Microbiol. 44,219-248 25. Mooser, G., Hefta, S., Paxton, R. J., Shirley, J. E., and Lee, T. D. (1991) J.

26. Blake, C. C. F., Johnson, L. N., Mair, G. A,, North, A. T. C., Phillips, D. C., and Sarno, V. R. (1967) Proc. R. Soc. London Ser. B. 167,37&385

27. Rodriguez, E. B. (1989) The Synthesis of Potential Inhibitors for Some Glucan Hvdrolases. Ph.D. thesis. DeDartment of Chemistrv. Universitv

Biol. Chem. 266,8916-8922

of Wester-n Australia, Nedlands,'Aus^tralia " _

28. Rodriguez, E. B., and Stick, R. V. (1990) Aut . J. Chem. 43,665-679 29. McCleary, B. V., and Glennie-Holmes, M. (1985) J. Imt. Brew. 9 1 , 285-

995 30. Sniteath. W. (1986) Bereev's Manual of Svstematic Bacterioloev. Vol. 2. D.

1236, Williams & Wil&, Baltimore " "l I

31. Anderson, M. A,, and Stone, B. A. (1975) FEBS Lett. 62, 202-207 32. Loo. J. A,. Udseth. H. R.. and Smith. R. D. (1989) Anal. Biochem. 179 .

A ~ A - A I i 33. Haj, P. B., Hartman, D. J., Morrice, N. A., Doan, D. N. P., and Fincher, G.

34. Zimmerman, C. L., Appella, E., and Pisano, J. J. (1977) Anal. Biochem. 7 7 ,

" _ "_ B. (1989) Plant Mol. Biol. 13,31-42

ux-vm 35. Hofemeister, J., Kurtz, A,, Borriss, R., and Knowles, J. (1986) Gene (Amst.)

"YY " I u

4 9 . 177-187 36. Murphy, N,-McConnell, D. J., and Cantwell, B. A. (1984) Nucleic Acids

Res. 12,5355-5367 37. Stone, B. A., and Clarke, A. E. (1993) Chemistry and Biology of U - 3 - p -

press Glucam, La Trobe University Press, Bundoora, Victoria, Australia, in

38. Woodward, J. R., and Fincher, G. B. (1982) Carbohydr. Res. 106,111-122 39. Fincher, G. B., Lock, P. A,, Morgan, M. M., Lingelbach, K., Wettenhall, R.

E. H.. Mercer. J. F. B.. Brandt. A,. and Thomsen. K. K. (1986) Proc. Natl. Acad. Sci. U. S. A.'83,2081-2085

40. Baird, S. D., Johnson, D. A., and Seligy, V. L. (1990) J. Bacteriol. 172 ,

41. Bueno, A., Vazquez de Aldana, C. R., Correa, J., and del Rey, F. (1990) 1576-1586

42. Baird, S. D., Hefford, M. A., Johnson, D. A,, Sung, W. L., Yaguchi, M., and Nucleic Acids Res. 18,4248

43. Rouvinen, J., Bergfors, T., Teeri, T., Knowles, J. K. C., and Jones, T. A. Seligy, V. (1990) Biochirn. Biophys. Res. Cornmun. 169 , 1035-1039

(1990) Science 249.380-385

, . Natl. Acad. Sci. U. S. A.'83,2081-2085

, .

40. Baird, S. D., Johnson, D. A., and Seligy, V. L. (1990) J. Bacteriol. 172 , 167G-1 KQG

41. Bueno, A., Vazquez de Aldana, C. R., Correa, J., and del Rey, F. (1990) A- ." *""

42. Baird, S. D., Hefford, M. A., Johnson, D. A,, Sung, W. L., Yaguchi, M., and Nucleic Acids Res. 18,4248

43. Rouvinen, J., Bergfors, T., Teeri, T., Knowles, J. K. C., and Jones, T. A. Seligy, V. (1990) Biochirn. Biophys. Res. Cornmun. 169 , 1035-1039

(1990) Science 249.380-385 44. Teathe6 R. M., and Erfle, J. D. (1990) J. Bacteriol. 172 , 3837-3841 45. Borriss, R., Buettner, K., and Maentsaelae, R. (1990) Mol. Gen. Genet.

46. Erfle, J. D., Teather, R. M., Wood, P. J., and Irvin, J. E. (1988) Biochem.

47. Huber, D. J., and Nevins, D. J. (1977) Plant Physiol. 60,300-304 48. Sierks, M. R., Ford, C., Reilly, P. J., and Svensson, B. (1990) Protein Eng.

49. Jeffcoat, R., and Kirkwood, S. (1987) J. Biol. Chem. 262 , 1088-1091

222,278-283

J. 266,833-841

3, 193-198

25066 Active-site Glutamate of B. amyloliquefaciens Endohydrolase 50. Tomme, P., Chauvaux, S., Beguin, P., Millet, J., Aubert, J.-P., and Claeys-

51. Weaver, L. H., and Matthews, B. W. (1987) J. Mol. Biol. 193,189-199 52. Keitel, T., Granzin, J., Simon, O., Borriss, R., Thomson, K. K., Wessner,

53. Matsuura, Y., Kusonoki, M., Wakado, H., and Kakudo, M. (1984) J.

54. Dinur, T., Osiecki, K. M., Legler, G., Gatt, S., Desnick, R. J., and Grabowski,

56. Hermans, M. M. P., Kroos, M. A., van Beeumen, J., Oostra, B. A., and 55. Quaroni, A., and Semenza,, G. (1976) J. Biol. Chem. 261,3250-3253

57. Simpson, R. J., Moritz R. L., Rubira M. R., and Nice, E. C. (1989) Anal.

sens, M. (1991) J. Biol. Chem. 266,10313-10318

H., Hohne, W., and Heinemann, U. (1991) J. Mol. Biol. 218, 703-704

Biochem. (Tokyo) 95,697-702

G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1660-1664

Reuser, A. J. J. (1991) J. Biol. Chem. 266,13507-13512

Biochem. 177,221-236

58.

59.

60.

61.

62.

63.

64.

65.

Lloberas J. Perez-Pons, J. A., and Querol, E. (1991) Eur. J. Biochem.

Schimming S Schwarz, W. H., and Staudenbauer, W. L. (1992) Eur. J.

Yahata, N. Watanabe T. Nakamura Y. Yamamoto, Y., Shuwsei, K., and

Schwarz W. H Schimmin S Ruckna el, K. P., Burgschwaiger, S., Kreil,

Fukumori, F., Kudo, T.,’Narahash~, b., and Monkoshl, K. (1986) J. Gen.

Boucher, F., Mollosoli, R., and Durand, S. (1988) Nucleic Acids Res. 16,

Gre inet, O., Chebrou, M.-C., and Beguin P. (1988) J. Bacteriol. 170,4582-

Planas, A,, Juncosa, M., Lloberas, J., and Querol, E. (1992) FEBS Lett.

197,9371343

Biochem.’204,13-19

Tanaka,H. (1990) den: (Amst.) Sd, li3-117

G., anh Stauaenbauer !A? L:’(19? dm (Amsf.) 63,23-30

Microbiol. 132 2329-2335

9874

4 b S

308, 141-145

Identification of glutamic acid 105 at the active site of Bacillus ...

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