TAILIEUCHUNG - Báo cáo khoa học: Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase fromStreptomyces 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 kJÆmol )1 ) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water⁄hydroxide molecule acts as the reaction nucleophile. | ễFEBS Journal Catalytic mechanism of SGAP a double-zinc aminopeptidase from Streptomyces griseus Yifat F. Hershcovitz1 Rotem Gilboa2 Vera Reiland2 Gil Shoham2 and Yuval Shoham1 1 Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology Technion-Israellnstitute of Technology Haifa Israel 2 Department of Inorganic Chemistry The Laboratory for StructuralChemistry and Biology The Hebrew University of Jerusalem Israel Keywords aminopeptidase catalytic mechanism catalytic residues fluoride inhibition isotope effect Correspondence Y. Shoham Department of Biotechnology and Food Engineering Technion Haifa 32000 Israel Fax 972 4 8293399 Tel 972 4 8293072 E-mail yshoham@ Received 30 April2007 revised 28 May 2007 accepted 1 June 2007 doi 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 with a Ki of mM at pH indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pKi experiments resulted in a pKa value of 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 whereas two proton transfers are implicated at pH . The role of Glu131 in binding and catalysis was assessed by .

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