1k89
From Proteopedia
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K89L MUTANT OF GLUTAMATE DEHYDROGENASE
Overview
Comparisons of the structures of glutamate dehydrogenase (GluDH) and, leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep, within the amino acid binding pockets of these homologous enzymes, from, hydrophilic residues to hydrophobic ones are critical components of their, differential substrate specificity. When one of these residues, K89, which, hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in, GluDH, was altered by site-directed mutagenesis to a leucine residue, the, mutant enzyme showed increased substrate activity for methionine and, norleucine but negligible activity with either glutamate or leucine. In, order to understand the molecular basis of this shift in specificity we, have determined the crystal structure of the K89L mutant of GluDH from, Clostridium symbiosum. Analysis of the structure suggests that further, subtle differences in the binding pocket prevent the mutant from using a, branched hydrophobic substrate but permit the straight-chain amino acids, to be used as substrates.The three-dimensional crystal structure of the, GluDH from C. symbiosum has been previously determined in two distinct, forms in the presence and absence of its substrate glutamate. A comparison, of these two structures has revealed that the enzyme can adopt different, conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved, in the hydride transfer reaction. It has previously been proposed that, this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical, conformation with that of the wild-type enzyme in the presence of, substrate. Comparison of the mutant structure with both the wild-type open, and closed forms has enabled us to separate conformational changes, associated with substrate binding and domain motion and suggests that the, domain closure may well be a property of the wild-type enzyme even in the, absence of substrate.
About this Structure
1K89 is a Single protein structure of sequence from Clostridium symbiosum. Active as Glutamate dehydrogenase, with EC number 1.4.1.2 Full crystallographic information is available from OCA.
Reference
Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum., Stillman TJ, Migueis AM, Wang XG, Baker PJ, Britton KL, Engel PC, Rice DW, J Mol Biol. 1999 Jan 15;285(2):875-85. PMID:9878450
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