Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase.
The replacement of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase by Asn and Gln by site-directed mutagenesis results in essentially complete inactivation of the enzyme, while replacement by Asp generates a mutant with a rate constant for the first step, formation of the glycosylenzyme, some 2500 times lower than that of the native enzyme. This low activity is shown to be a true property of the mutant and not due to contaminating wild-type enzyme by active site titration studies and also through studies of its thermal denaturation and of the pH dependence of the reaction catalyzed. Binding of ground-state inhibitors is affected relatively little by the mutation, while binding of transition-state analogues is greatly impaired, consistent with a principal role for Glu 358 being in transition-state stabilization, not substrate binding. Determination of kinetic parameters for a series of aryl glucosides revealed that the glycosylation step is rate determining for all these substrates in contrast to the native enzyme, where a switch from rate-limiting glycosylation to rate-limiting deglycosylation was observed as substrate reactivity was increased. These results coupled with secondary deuterium kinetic isotope effects of kH/kD = 1.17 and 1.12 measured for the 2,4-dinitrophenyl and p-nitrophenyl glucosides point to a principal role of the nucleophile in stabilizing the cationic transition states and in formation of the covalent intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, University of British Columbia, Vancouver, Canada.
S G
SG
Withers
K
K
Rupitz
D
D
Trimbur
R A
RA
Warren
D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites / Q000235:genetics; D003903:Deuterium; D004355:Drug Stability; D005960:Glucosides / Q000378:metabolism; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D005973:Glutamine; D006031:Glycosylation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000235:genetics*
D005960:Glucosides; D005971:Glutamates; D005973:Glutamine; C070512:2',4'-dinitrophenylglucopyranoside; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D003903:Deuterium; D001617:beta-Glucosidase