1alh
From Proteopedia
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KINETICS AND CRYSTAL STRUCTURE OF A MUTANT E. COLI ALKALINE PHOSPHATASE (ASP-369-->ASN): A MECHANISM INVOLVING ONE ZINC PER ACTIVE SITE
Overview
Using site-directed mutagenesis, an aspartate side chain involved in, binding metal ions in the active site of Escherichia coli alkaline, phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and, by alanine (D369A). The purified mutant enzymes showed reduced turnover, rates (kcat) and increased Michaelis constants (Km). The kcat for the, D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme., The D369N enzyme required Zn2+ in millimolar concentrations to become, fully active; even under these conditions the kcat measured for hydrolysis, of p-nitrophenol phosphate was 2 orders of magnitude lower than for the, wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50, times less efficient when the carboxylate side chain of Asp-369 is, replaced by the corresponding amide; and activity is reduced to near, nonenzymic levels when the carboxylate is replaced by a methyl group. The, crystal structure of D369N, solved to 2.5 A resolution with an R-factor of, 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of, the kinetic results and the refined X-ray coordinates, a reaction, mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme, involving only 1 metal with the possible assistance of a histidine side, chain.
About this Structure
1ALH is a Single protein structure of sequence from Escherichia coli with ZN, PO4 and SO4 as ligands. Active as Alkaline phosphatase, with EC number 3.1.3.1 Full crystallographic information is available from OCA.
Reference
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site., Tibbitts TT, Xu X, Kantrowitz ER, Protein Sci. 1994 Nov;3(11):2005-14. PMID:7703848
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