1KBK

Mechanistic Analyses of Catalysis in Human Pancreatic Alpha-Amylase: Detailed Kinetic and Structural Studies of Mutants of Three Conserved Carboxylic Acids

Summary for 1KBK

• Entry DOI: 10.2210/pdb1kbk/pdb
• Descriptor: ALPHA-AMYLASE, PANCREATIC, 2-acetamido-2-deoxy-beta-D-glucopyranose, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: amylase, glycosylation, mutagenesis, diabetes, hydrolase, glycosidase, carbohydrate metabolism, catalysis, pancreatic, enzyme, human
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 56184.03

Authors

Rydberg, E.H.,Li, C.,Maurus, R.,Overall, C.M.,Brayer, G.D.,Withers, S.G. (deposition date: 2001-11-06, release date: 2002-04-10, Last modification date: 2021-10-27)

Primary citation

Rydberg, E.H.,Li, C.,Maurus, R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
Biochemistry, 41:4492-4502, 2002

PubMed Abstract: The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to 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 mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) 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 at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.

PubMed: 11914097
DOI: 10.1021/bi011821z

Experimental method

X-RAY DIFFRACTION (1.9 Å)