Tài liệu Báo cáo khoa học: Catalytic mechanism of SGAP, a double-zinc aminopeptidase from

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-Israel Institute of Technology, Haifa, Israel

2 Department of Inorganic Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel

Aminopeptidases are exopeptidases that catalyze the

removal of N-terminal amino acids from peptides; they

are found in bacteria, plants and mammalian tissues.

Many aminopeptidases are metallo-enzymes, containing

two catalytic transition metals (usually zinc) in their act￾ive site [1–3]. The activity of these enzymes is associated

with many central biological processes, such as protein

maturation, protein degradation, hormone level regula￾tion, angiogenesis and cell-cycle control [4–8]. Not

surprisingly, aminopeptidases play an important role in

many pathological conditions, including cancer, cata￾ract, cystic fibrosis and HIV infection. Indeed, anti￾tumor drugs such as ovalicin and fumagillin were found

to inhibit aminopeptidases. In this regard, the natural

inhibitor for aminopeptidases, bestatin, was recently

shown to significantly decrease HIV infection by inhibit￾ing aminopeptidase activity [9–11]. Aminopeptidases

can be classified into clans and families based on their

amino acid sequence homology. Clan M contains

mainly metallopeptidase families, one of which is M28.

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: [email protected]

(Received 30 April 2007, revised 28 May

2007, accepted 1 June 2007)

doi:10.1111/j.1742-4658.2007.05912.x

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 kJÆmol)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 non￾competitive inhibitor of SGAP at pH values of 5.9–8 with a Ki 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 pKi experi￾ments resulted in a pKa 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 (Km, kcat) over a tempera￾ture range of 293–329 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 resul￾ted in a four orders of magnitude decrease in kcat, corresponding to an

increase of 9 kJÆmol)1 in the activation energy for the E131D mutant,

emphasizing the crucial role of Glu131 in catalysis.

Abbreviations

AAP, Aeromonas proteolytica aminopeptidase; blLAP, bovine lens leucine aminopeptidase; Leu-pNA, leucine-para-nitroanilide; SGAP,

Streptomyces griseus aminopeptidase.

3864 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS