Steady-State Kinetic Characterization of Substrates and Metal-Ion Specificities of the Full-Length and N-Terminally Truncated Recombinant Human Methionine Aminopeptidases (Type 2)
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- 作者：Guang Yang ; Robert B. Kirkpatrick ; Thau Ho ; Gui-Feng Zhang ; Po-Huang Liang ; Kyung O. Johanson ; David J. Casper ; Michael L. Doyle ; Joseph P. Marino ; Jr. ; Scott K. Thompson ; Wenfang Chen ; David G. Tew
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：September 4, 2001
- 年：2001
- 卷：40
- 期：35
- 页码：10645 - 10654
- 全文大小：110K
- 年卷期：v.40,no.35(September 4, 2001)
- ISSN：1520-4995
文摘
The steady-state kinetics of a full-length and truncated form of the type 2 human methionineaminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of variouspeptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA),utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assaymethod. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acidsfor which the values of kcat/Km were approximately 5 × 105 M-1 min-1. It was found that while thenature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2,the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptidesubstrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminishedcatalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almostentirely due to the reduction in the turnover rate (kcat), suggesting that cleavage of the amide bond is atleast partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either thepeptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamicanalyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kineticbehavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activatethe full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 underany concentration that was tested.