Human muscle glyceraldehyde-3-phosphate dehydrogenase: reactivity of sulphydryl groups.

1. The kinetics of the reaction of thiol groups of glyceraldehyde-3-phosphate dehydrogenase from human muscle with 5,5'-dithiobis-2-nitrobenzoate (DTNB) was studied spectrophotometrically using the conventional and stopped-flow methods. 2. Each of the three thiol groups present in the enzyme subunit reacts with a different velocity. 3. The reaction with DTNB of the four sulphydryl groups of Cys-149, essential for the enzymatic activity, is biphasic, depends on the amount of NAD bound and is strongly slowed down when one mole of coenzyme is bound to the tetramer. NAD bound is only partially released from the holoenzyme treated with DTNB. 4. In the presence of borate, thiol groups of Cys-153 and Cys-244 do not react with DTNB.