title: 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 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.
authors.affiliation: Institut für Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany. strater@chemie.fu-berlin.de
authors.forename: N
authors.initials: N
authors.lastname: Sträter
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
authors.forename: W N
authors.initials: WN
authors.lastname: Lipscomb
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry; D001639:Bicarbonates / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D043384:Glutamyl Aminopeptidase; D006868:Hydrolysis; D007931:Leucyl Aminopeptidase / Q000737:chemistry*; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D001639:Bicarbonates; D010455:Peptides; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; D043384:Glutamyl Aminopeptidase