A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

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 Å and 1.6 Å 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 inf luences 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.

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

“…The metals may help orient SLG and stabilize the excess negative charge formed in a tetrahedral transition state. The participation of metal ions in orienting substrate for nucleophilic attack has been proposed in several other binuclear metalloenzymes, including the metallo-␤-lactamases and leucine aminopeptidase (33,38,40). Once SLG has been bound and oriented for nucleophilic attack, the hydroxide, now bound only to metal 2 and held in position by Asp-58, can attack the lactoyl carbonyl.…”

“…In leucine aminopeptidase, a lysine residue homologous to Lys-142 of glyoxalase II has been shown to participate in transition state stabilization (40). Replacing this lysine with alanine in leucine aminopeptidase resulted in a 99% reduction in activity (40).…”

“…Many binuclear metalloenzymes, such as the metallo-␤-lactamases and leucine aminopeptidase, appear to utilize a hydroxide, arising from a bridging water molecule in the nucleophilic attack of substrate (33,40). Residues that hydrogen-bond to the nucleophilic hydroxide usually play a critical role in orienting the hydroxide and holding it in a fixed position that reduces the entropic barrier for nucleophilic attack on the substrate (41).…”

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

“…X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al, 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al, 1999b;Gu and Walling, 2002).In most plants, three classes of LAP-related polypeptides are detected using a tomato LAP antiserum, including the 66-and 77-kD LAP-like proteins and the 55-kD neutral LAP (LAP-N; Chao et al, 2000). Only in a subset of the Solanaceae is a second 55-kD LAP species (LAP-A) detected (Hildmann et al, 1992;Gu et al, 1996b;Chao et al, 2000).…”

“…X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al, 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al, 1999b;Gu and Walling, 2002).…”