Ágoston Hoschke

A study by means of lactone inhibition of the role of a “half-chair” glycosyl conformation at the active centre of amylolytic enzymes

Abstract The kinetics of inhibition of porcine-pancreatic alpha amylase, sweet-potato beta amylase, and Aspergillus niger glucamylase enzymes have been studied by use of d -glucono-l,5-lactone and maltobiono-1,5-lactone as transition-state analogs. With d -glucono-1,5-lactone, alpha amylase can be inhibited, to a degree, non-competitively (K i 0.81m M ,β≈e0.2), whereas with maltobionolactone, the inhibition is competitive (K i 10.31m M ). The effect of beta amylase can be inhibited with maltobionolactone in a completely competitive way (K i O.11m M ), whereas with d -gluconolactone the inhibition is very poor (K i 21m M ). Glucoamylase cannot be inhibited with maltobionolactone, whereas with d -gluconic acid, a completely mixed inhibition way be observed (K i 1.3m M ). The ratio of the binding affinity of the lactones, products, and substrates, permits the conclusion that ring distortion takes place in the transition state with all three enzymes.

A study of the role of histidine side-chains at the active centre of amylolytic enzymes☆

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.

The biosynthesis of starch : Part XIII. Inhibition of potato phosphorylase by donor substrate analogues☆☆☆

Abstract The inhibitory effect of d -glucose, 2-deoxy- d - arabino -hexose, 3- O -methyl- d -glucose, and 6- O -methyl- d -glucose on the enzymic synthesis catalysed by potato phosphorylase has been investigated. Kinetic methods showed that, for both of the substrates employed, a competitive inhibitor effect was exerted by each of the substrate analogues examined. Values for K i and K m calculated from the experimental data indicate that, in the presence of each of the added substrate analogues, the affinity of the enzyme for both substrates decreased. On the basis of the changes in K i values, it appeared that, in the case of each substrate, the inhibitor effect of d -glucose was suppressed by the absence of the hydroxyl groups at the positions 2 and 3.

Biosynthesis of starch : Part XI. Inhibition effects of thiol reagents on phosphorylase action☆

Abstract The inhibition effects of the reagents p-chloromercuribenzoate (pCMB), iodoacetamide (IAA), and N-ethylmaleimide (NEMI) in the reaction catalysed by phosphorylase were investigated. The dissociation constants of the complexes of the various inhibitors formed with the enzyme were established. The values of Ki were 4.4 x 10−6 (pCMB), 1.2 x 10−2 (IAA), and 8.7 x 10−2 (NEMI). It was thus inferred that latent SH groups play an essentially greater role than do free SH groups. Although it is unlikely that they participate in the formation of the enzyme-substrate complex, they presumably stabilise a definite enzyme orientation. It was found that the reagents tested cause irreversible inhibition, and that the substrates exert a protecting effect.