Chemical modification of octopine dehydrogenase by thiol-specific reagents: evidence for the presence of an essential cysteine at the catalytic site.
Thiol-specific reagents, p-chloromercuricphenyl sulfonic acid (PCMS), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethyl-maleimide (NEM), incubated with octopine dehydrogenase resulted in the loss of catalytic activity. The kinetic profile of PCMS inactivated enzyme showed that the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. The reversal of enzyme activity was obtained by thiol-containing reagents. The loss of enzyme activity was prevented only by NADH and not by other substrates. The dissociation constant for NADH calculated from the decrease in the enzyme inactivation rate was 45 microM. Cyanolysis of the DTNB-modified enzyme with KCN led to the release of 5-thio-2-nitrobenzoate (TNB) accompanied by the formation of thio-cyano enzyme. By correlating the enzyme activity with the formation of thio-cyano derivative it was found that no activity was recovered after KCN treatment. These evidences clearly established the critical involvement of the thiol group in catalysis. Double inhibition studies with PCMS and NEM on octopine dehydrogenase showed that the inactivating reagents bind to the same functional thiol present at the catalytic center. pH-dependent inactivation by PCMS indicated that a group with a pKa value of 7.4 is involved in the loss of enzyme activity. These approaches suggested that at least one thiol group present at the coenzyme-binding domain is essential for the catalytic reaction of octopine dehydrogenase.
Department of Chemistry, Indian Institute of Technology, Kanpur.
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S
Sheikh
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Katiyar
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry* / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D009243:NAD; D000594:Amino Acid Oxidoreductases; C021903:D-octopine dehydrogenase; D003545:Cysteine