Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus.
The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 +/- 5 kJ.mol(-1)) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9-8 with a K(i) of 11.4 mM at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pK(i) experiments resulted in a pK(a) value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid-base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K(m), k(cat)) over a temperature range of 293-329 degrees K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in k(cat), corresponding to an increase of 9 kJ.mol(-1) in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.
Hershcovitz
Yifat F
YF
Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa, Israel.
Gilboa
Rotem
R
Reiland
Vera
V
Shoham
Gil
G
Shoham
Yuval
Y
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium; D004795:Enzyme Stability; D005461:Fluorine / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology; D012997:Solvents; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D007477:Ions; D010710:Phosphates; D012997:Solvents; D005461:Fluorine; D003903:Deuterium; D000626:Aminopeptidases; D015032:Zinc