Alkaline phosphatase Structure and biophysics
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Transcript of Alkaline phosphatase Structure and biophysics
Alkaline Phosphatase (AP)Presented by: Rimjhim Roy Choudhury
•Hydrolase enzyme: Removal or transfer of phosphoryl groups•Metalloenzymes•Most effective in an alkaline environment.•A 2-fold symmetric dimer (100 Å x 50 Å x 50 Å ), 449 amino acids per monomer• 2 active sites located about 30 Å from each other.•Class: Alpha and beta proteins (α/β) •Mainly parallel beta sheets (beta-alpha-beta units)•Fold: Alkaline phosphatase-like • Core:• 3 layers: α/β/α• mixed beta-sheet of 8 strands, order 43516728, strand 7 is antiparallel to the rest
Structure
• Accepted kinetic scheme for the enzymatic hydrolysis/transphosphorylation of phosphate monoesters by AP (Scheme 1) •E-P : Phosphoseryl enzyme formed with Ser102•E. ROP or E . P : Noncovalent complexes with substrate or product. •The hydrolysis of E-P involves both formation and dissociation of the non-covalent enzyme-phosphate complex (E . P). •If a phosphate acceptor such as ethanolamine or Tris is present in high concentration, then the enzyme transfers the phosphate from the substrate to the alcohol (R2OH)
Kinetic Scheme
Active Site
•Ribbon drawing of a monomer of alkaline phosphatase•Each active site of the dimeric enzyme contains three metal-binding sites (M1, M2 and M3) which consists of metal triplet (two Zn2+ and one Mg2+) and their ligands•It consists of a 10-stranded central β-sheet flanked by 15 helices and another 3-stranded β-sheet, and a helix on the top.•This site is located at the carboxyl end of the central β-sheet and all the ligands to the three metal ions are provided from one monomer.•The 3 metals are shown as stippled spheres.
Geometry around metals in phosohate-bound AP (subunit A/ subunit B)
•The active site region can be considered as Asp101-Ser102-Ala103 and the metal triplet (two Zn2+ and one Mg2+) and their ligands, as well as Arg166 and other amino acids in the immediate vicinity. •The tetrahedral phosphate participates in interactions with the two active-site zinc ions, the guanidino nitrogen atoms of Arg166 and two water molecules. •The bidentate interaction of Arg166 with the phosphate oxygen atoms is planar.•The catalytic metal triad is similar to that of phospholipase C from Bacillus cereus and P1 nuclease from Penicillium citrinum
The Catalytic Triad
(M1)
(M2)
(M3)
Three Metal Ions Catalysis
•The zinc atom at M2 activates Ser102, which performs the initial attack on the phosphate group of the substrate. •The zinc atom at M1 activates a water molecule, which is well positioned for the apical attack of the phosphoseryl intermediate.•Study by Stec et al. showed that Ser102 is orientated in an activating conformation toward the zinc ion at M2 only when magnesium is present at M3. •In addition, the octahedral geometry of the magnesium ion at M3 properly positioned a Mg-bound water molecule that plays the role of a general base in the generation of the Ser102 nucleophile, and of a general acid in the regeneration of the Ser102 hydroxyl group.•Asp153 is a key residue in the stabilisation of the magnesium ion at M3.
(M2)
(M1)
(M3)
(M2)
(M1)
(M3)
(M2) (M2)
(M1) (M1)
(M3) (M3)
Importance
Bacteria:•Generate free phosphate groups for uptake and use.•Helps in uptake of organic compounds in the wildResearch:Molecular biology laboratories.•Prevents DNA ligation, thereby keeping DNA molecules linear.•Allows radiolabeling for measuring the presence of the labelled DNA. •Enzyme immunoassays•Thermolabile Alkaline phosphatase from shrimp is the most usefulHumans:Alkaline phosphatase isozymes:ALPI – intestinalALPL – tissue non-specific (liver/bone/kidney)ALPP – placental (Regan isozyme)Diagnostic useNormal range is 20 to 140 IU/LElevated levels•Jaundice and hepatitis•Isoenzyme studies using electrophoresis can confirm the source of the ALP. Lowered levels• Hypophosphatasia. •Aplastic anemia, pernicious anemia, children with cretinism•Levels have been observed to increase in men who have undergone heart surgery
Structural alignment generated by pairwise comparison of the structures of ECAP, TAP, SAP, and PLAP
Blue stars indicate residues involved in metal binding or being involved in catalysis and specificity.•AP from E. coli (ECAP)•AP from Human placenta (PLAP) •AP from Shrimp (SAP)•AP from Antarctic Bacterium (TAP)
M. de Backer, S. McSweeney, H.B. Rasmussen, B.W. Riise, P. Lindley, E. Hough, The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase, J. Mol. Biol. 318 (2002) 1265–
1274.
•The enzyme is used extensively in vitro to dephosphorylate DNA or dNTPs•Can be inactivated by a short rise in temperature to 65˚C.•Species that live in such environments must somehow be adapted to survive at these reduced temperatures.•A lower temperature has a number of implications for the functioning of proteins. •The increased viscosity of water, which reduces the diffusion rates of substrate and product, leads to lower reaction rates.•Reduced thermal motions affect the enzyme’s overall stability, kinetics and ligand binding.
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Cold Adaptation
Cold-active enzymes have :•An increased catalytic efficiency at lower temperature, •A reduced thermal stability•Increased molecular flexibility.
Features:•APs usually contain a magnesium ion in the third metal-binding site, while a zinc ion is observed in SAP.•The arginine residue in SAP (Arg162) corresponding to ECAP Arg166 is oriented differently; it points away from the metal ions •Modification of the arginine residue leads to a lower substrate affinity, but not to a significant decrease of the activity.•SAP has a surplus of negatively charged amino acid residues and a relatively low number of proline residues, while the frequency of aromatic residues is higher
•The sequences of SAP and ECAP have 24% identical, 16% strongly similar and 11% weakly similar residues. •SAP and PLAP share 41% of their residues, 19% strongly•similar and 12% weakly similar. •The active site residues are conserved among these three sequences, except for three residues; Asp153 and Lys328 are histidines in SAP (His149 and His316) and PLAP (His153 and His317), and PLAP has a T155S substitution (ECAP-Thr155, SAP-Thr151, PLAP-Ser155)
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•The overall charge of -80 for the functional dimer is distributed such that the protein surface is predominantly negatively charged. •The active site is the only clear positively charged patch on the surface. •As a result, the negatively charged substrate (a phosphomonoester) must be strongly directed towards the active site.
Surface potential representation
Cold-adaptation in SAP was largely traced to extensive negative potential on its surface
•Appleburry, M. L., Johnson, B. P. & Coleman, J. E. (1970). Phosphate binding to alkaline phosphatase. J. Biol. Chem. 245, 4968-4976.•Kim, E. E. & Wyckoff, H. W. (1991). Reaction mechanism of alkaline phosphatase based on crystal structures two metal-ion catalysis. J. Mol. Biol. 218, 449-464.•Kathleen M. Holtz, Evan R. Kantrowitz (1999). The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis. FEBS Letters 462, 7-11•Boguslaw Stec, Kathleen M. Holtz & Evan R. Kantrowitz (2000). A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J. Mol. Biol. 299, 1303-1311•M. de Backer, S. McSweeney, H.B. Rasmussen, B.W. Riise, P. Lindley, E. Hough (2002). The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase. J. Mol. Biol. 318, 1265–1274.
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