[Sulfhydryl groups of alpha-ketoglutarate dehydrogenase from pigeon breast muscle].
Using various thiol reagents (DTNB, pCMB, NBD-chloride), the number and reactivity of SH-groups of the alpha-ketoglutarate dehydrogenase component (KGD) of the alpha-ketoglutarate dehydrogenase complex were established. The total number of SH-groups as determined in the presence of a detergent is 12 per monomer of KGD. In the native enzyme DTNB titrates 3 or 4 SH-groups which, according to their reactivity, can be divided into 3 types. pCMB and NBD-chloride modify 5 to 6 SH-groups of KGD. In the presence of alpha-ketoglutarate and its active analog, alpha-ketoadipate, two SH-groups of the enzymes, the most and the least reactive ones, become inaccessible to the action of DTNB but the titrated by a 30-100-fold molar excess of pCMB. Binding of alpha-ketoacids which are competitive inhibitors of the enzyme causes no masking of SH-groups of KGD. When 3-4 SH-groups of KGD are blocked, the enzyme activity is reduced by 30% due to modification of the same two SH-groups which are inaccessible to DTNB in the presence of substrate. The fluorescence spectra of S-NBD derivatives of KGD suggest that these two SH-groups have hydrophobic environment. In terms of the number and reactivity of the thiol groups the apoenzyme does not differ from the holoenzyme; however, an addition of alpha-ketoglutarate to the apoenzyme does not decrease the number of SH-groups interacting with DTNB or the rate of their modification. The masking of two SH-groups of the holoenzyme results from conformational changes accompanying the formation of a productive ternary enzyme -- coenzyme -- substrate complex. Possible localization of these groups in the proximity of the active site is discussed.
D000818:Animals; D010856:Columbidae; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex
Sul'fgidril'nye gruppy al'fa-ketoglutaratdegidrogenazy iz grudno myshtsy golubia.