Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure.
Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents. This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 236, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity. The p-CMB inactivation could be largely reversed on the addition of excess cysteine. A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme.
Malhotra
O P
OP
Department of Chemistry, Banaras Hindu University, Varanasi.
Srivastava
D K
DK
Kayastha
A M
AM
Srinivasan
D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007700:Kinetics; D010944:Plants / Q000201:enzymology; D010946:Plants, Medicinal; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013552:Swine; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D013439:Sulfhydryl Reagents; D020245:p-Chloromercuribenzoic Acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005033:Ethylmaleimide