A study of the role of histidine side-chains at the active centre of amylolytic enzymes |
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Authors: | Ágoston Hoschke Elemér László János Holló |
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Affiliation: | Department of Agricultural Chemical Technology, Technical University Budapest, H1521 Budapest, Gellért tér 4 Hungary |
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Abstract: | The role of histidine side-chains in reactions catalysed by porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase has been studied by using diethyl pyrocarbonate, a specific protein reagent. Changes in the activity, binding affinity, and apparent kinetic parameters due to ethoxycarbonylation have been determined. For pancreas alpha-amylase, four of the eight histidine groups, for sweet-potato beta-amylase, six of the seven histidine groups, and for A. niger glucamylase, four of the six histidine groups were shown to be ethoxycarbonylated. Ethoxycarbonylation occurred as an apparent first-order reaction, with rate constants in the range 3.6–4.9 x 10?2min?1. Ethoxycarbonylation of the histidine group at the active centre rapidly inactivated alpha-amylase, whereas the other three groups are not located in the active centre, although activity and substrate binding are only slightly affected by their modification. For beta-amylase and glucamylase, only slight or no change in activity could be detected on ethoxycarbonylation, whereas significant changes were observed in the binding affinity. |
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