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The Three-Dimensional Structure of Asn102 Mutant of Trypsin : Role of Asp102 in Serine Protease Catalysis S. Sprang, T. Standing, R. J. Fletterinck, R. M. Stroud, J. Finer-Moore, N-H. Xuong, R. Hamlin, W. J. Rutter, C. S. Craik The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.

Fig. 3. (A) In the hydrogen bond network found in D 102 N trypsin above neutral pH, His57 is unable to accepts a proton from Ser195 Oδ. The orientation of the hydrogen bond between His57 and Ser195 is the reverser of that observed in the bovine trypsin-benzamindine structure (7). (B) In the hydrogen bond network of wild-type trypsin, His57 is an acceptor for the proton from Ser195.

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Figure 1. Top : A MacImdad (Molecular Applications Group, Stanford, CA) drawing of the refined coordinates of the active site of trypsin D102S/S214D shown with the His 57 imidazolium nitrogens labeled and shaded. The hydrogen bonds and distances of interest are identified. Leu 99 is a surface residue whose side chain is responsible, in part, for the solvent inaccessibility of the amino acids at positions 102 and 214. Bottom : A MacImdad drawing of the active sites of trypsin and trypsin D102S/S214D shown superimposed by Cα. Trypsin D102S/S214D, which features Ser 102, His 57, Ser 195, and Asp 214, is represented in thick lines, while trypsin is shown in thin lines. Note that trypsin His 57 Nε2 forms a long hydrogen bond with Ser 195 Oγ, while in trypsin D102S/S214D His 57 N δ 1 is the proximal nitrogen.

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Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin.

BY J. A. WELLS1, B. C. CUNNINGHAM2 AND D. A. ESTELL2

Department of Biocatalysis, Genentech, Inc., 460 Point San Bruno Boulevard,

South San Francisco, California 94080, U.S.A. Research Department, Genencor, Inc., 180 Kimball Way,South San Francisco, California 94080, U.S.A.

Structural studies on serine proteinases have shown that hydrogen bonds are involved in stabilizing the charged tetrahedral intermediate in the transition-state complex. However, little is known about the quantitative contribution of these interactions to transition-state stabilization. X-ray crystallographic studies of subtilisin (Robertus, J. D., Kraut, J., Alden, R. A. & Birktoft, J. J. Biochemistry, Wash. 11, 4293-4303 (1972)) have suggested that the amide side chain from asparagines-155 forms a Hydrogen bond with the oxyanion produced on the substrate carbonyl oxygen in the tetrahedral intermediate. To study the importance of the Asn-155 hydrogen bond in stabilizing the tetrahedral intermediate, Asn-155 was substituted with Thr, His, Gln19 and Asp by using site-specific mutagenesis of the cloned subtilisin gene from B. amyloliquefaciens. These substitutions were intended to alter the position and charge of the potential hydrogen-bonding group at 155. Mutations of Asn-155 caused large decreases in substrate turnover, kcat (200-to 4000-fold), with marginal decreases in substrate binding, KM (up to 7-fold). The most dramatic effects were seen with Thr-155, where kcat was reduced 4000-fold with a slight increase in KM. Mutations of Asn-155 caused a loss in transition-state stabilization energy of 9.2-20 kJ mol-1.

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Paramètres cinétiques et diagramme d’énergie

E S ES E PKs

kcat/KM

+

+ +E S E

+

P

ES

E S+

Gs

Gcat

ES++

G++

kcat

L’enzyme accélère la vitesse d’une réaction en stabilisant l’état de transition.

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