Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamtHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use...

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Bibliographic Details
Published in:Protein engineering 1990-05, Vol.3 (6), p.515-521
Main Authors: Minard, P., Bowen, D.J., Hall, L., Littlechild, J.A., Watson, H.C.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Aspartic Acid - genetics
ATP
ATP binding
Base Sequence
Binding Sites
Escherichia coli - genetics
Gene Expression
Genetic Vectors
glycolytic enzyme
Kinetics
Molecular Sequence Data
Mutation
Phosphoglycerate Kinase - genetics
Protein Conformation
protein engineering
Saccharomyces cerevisiae
site-directed mutagenesis
Yeasts - enzymology
Yeasts - genetics
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Summary:A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamtHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystaUographic and NMR experiments. Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK. The function of this acidic residue appears to be to help desolvate the magnesium ion compfexed with either ADP or ATP when this substrate binds to the enzyme. Both crystallographk and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure. The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phospho-glycerate. The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N). This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme. This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.
ISSN:1741-0126
0269-2139
1741-0134
DOI:10.1093/protein/3.6.515