Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein.
The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid beta-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k(cat), with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.
Thompson
Michael W
MW
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0298, USA.
Govindaswami
Meera
M
Hersh
Louis B
LB
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004399:Dynorphins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D014443:Tyrosine; D011691:Puromycin; D004399:Dynorphins; D000626:Aminopeptidases