PMID:10500145
| Citation |
Sträter, N, Sun, L, Kantrowitz, ER and Lipscomb, WN (1999) A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase. Proc. Natl. Acad. Sci. U.S.A. 96:11151-5 |
|---|---|
| Abstract |
The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold. |
| Links | |
| Keywords |
Aminopeptidases/chemistry; Bicarbonates/pharmacology; Binding Sites; Catalysis; Enzyme Activation; Glutamyl Aminopeptidase; Hydrolysis; Leucyl Aminopeptidase/chemistry; Mutation; Peptides/metabolism; Static Electricity; Structure-Activity Relationship |
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