Mechanistic Analyses of Catalysis in Human Pancreatic -Amylase: Detailed Kinetic and Structural Studies of Mutants of Three Conserve
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- 作者：Edwin H. Rydberg ; Chunmin Li ; Robert Maurus ; Christopher M. Overall ; Gary D. Brayer ; and Stephen G. Withers
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：April 2, 2002
- 年：2002
- 卷：41
- 期：13
- 页码：4492 - 4502
- 全文大小：148K
- 年卷期：v.41,no.13(April 2, 2002)
- ISSN：1520-4995
文摘
The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalyticmechanism of human pancreatic 
-amylase (HPA) were studied by utilizing site-directed mutagenesis incombination with structural and kinetic analyses of the resultant enzymes. All three residues were mutatedto both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated.Structural analyses demonstrated that there were no significant differences in global fold for the mutantenzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All resultssuggested that D197 was the nucleophile, as virtually all activity (>105-fold decrease in kcat values) waslost for the enzymes mutated at this position when assayed with several substrates. The significantlygreater second-order rate constant of E233 mutants on "activated" substrates (kcat/Km value for -maltotriosylfluoride = 15 s-1 mM-1) compared with "unactivated" substrates (kcat/Km value for maltopentaose =0.0030 s-1 mM-1) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles.Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated atD300. At the least, this suggests an overall impairment of the catalytic mechanism where the reactionthen proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest thatD300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of theglycosidic bond.
-amylase (HPA) were studied by utilizing site-directed mutagenesis incombination with structural and kinetic analyses of the resultant enzymes. All three residues were mutatedto both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated.Structural analyses demonstrated that there were no significant differences in global fold for the mutantenzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All resultssuggested that D197 was the nucleophile, as virtually all activity (>105-fold decrease in kcat values) waslost for the enzymes mutated at this position when assayed with several substrates. The significantlygreater second-order rate constant of E233 mutants on "activated" substrates (kcat/Km value for -maltotriosylfluoride = 15 s-1 mM-1) compared with "unactivated" substrates (kcat/Km value for maltopentaose =0.0030 s-1 mM-1) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles.Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated atD300. At the least, this suggests an overall impairment of the catalytic mechanism where the reactionthen proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest thatD300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of theglycosidic bond.