TAILIEUCHUNG - Báo cáo khoa học: Human salivary a-amylase Trp58 situated at subsite )2 is critical for enzyme activity

The nonreducing end of the substrate-binding site of human salivarya-amylase contains two residues Trp58 and Trp59, which belong tob2–a2 loop of the catalytic (b/a)8 barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150–180-fold lower than the wild type) with starch as substrate | Eur. J. Biochem. 271 2517-2529 2004 FEBS 2004 doi Human salivary a-amylase Trp58 situated at subsite -2 is critical for enzyme activity Narayanan Ramasubbu1 Chandran Ragunath1 Prasunkumar J. Mishra1 Leonard M. Thomas2 Gyongyi Gyemant3 and Lili Kandra3 1 Department of Oral Biology University of Medicine and Dentistry of New Jersey Newark NJ USA 2Howard Hughes Medical Institute Division of Biology California Institute of Technology Pasadena CA USA 3Department of Biochemistry Faculty of Sciences University of Debrecen Hungary The nonreducing end of the substrate-binding site of human salivary a-amylase contains two residues Trp58 and Trp59 which belong to b2 a2 loop of the catalytic b a 8 barrel. While Trp59 stacks onto the substrate the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity 150-180fold lower than the wild type with starch as substrate. With oligosaccharides as substrates a reduction in kcat anincrease in Km and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides in contrast wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2 -3 and 2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived .

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