REACTIVITY OF SULFHYDRYL GROUPS OF LOBSTER MUSCLE GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE

Crystalline glyceraldehyde 3-phosphate dehydrogenase from lobster muscle was found to contain 19.5 ± 0.5 cysteine residues/140,000 g of enzyme. This estimate is based on titrations of native and modified forms of the enzyme in 8 M urea with 5,5′-dithiobis-(2-nitrobenzoic acid). Under nondenaturing conditions, in the presence of a40-fold molarexcess of 5,5′-dithiobis-(2-nitrobenzoic acid), all of the SH groups react within approximately 15 min; 4.5 ± 0.5 of these, i.e., 1 SH group/polypeptide chain, react immediately. Disulfide formation at the four, exceedingly reactive, SH groups results in complete inactivation of the enzyme. Carboxymethylation of the “active site” cysteine residues reduces the number of SH groups reactive toward 5,5′-dithiobis(2-nitrobenzoic acid) to 4.5 ± 0.5; approximately 12 SH groups are totally unreactive. Enzyme treated with iodosobenzoic acid, resulting in the formation of an intramolecular disulfide bond at the “active center” (Davidson, B. E., Sajgò, M., Noller, H. F., and Harris, J. I. (1967), Nature 216, 1181), has 11.0 ± 0.5 SH groups which react with 5,5′-dithiobis(2-nitrobenzoic acid). The steps which lead to the unmasking of all the SH groups of lobster muscle glyceraldehyde 3-phosphate dehydrogenase upon reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) are discussed. © 1969, American Chemical Society. All rights reserved.