Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding.
1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.
D000478:Alkylation; D000577:Amides; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors; D008961:Models, Structural; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D010070:Oxalates; D011758:Pyrroles; D011773:Pyruvates; D013438:Sulfhydryl Compounds; D013447:Sulfites; D013552:Swine
D000577:Amides; D010070:Oxalates; D011758:Pyrroles; D011773:Pyruvates; D013438:Sulfhydryl Compounds; D013447:Sulfites; D009243:NAD; D007770:L-Lactate Dehydrogenase