The Properties of Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase Labeled with a "Reporter Group"

Abstract

Abstract A group, 2-acetamide-4-nitrophenol, has been incorporated at the active site sulfhydryl group of each of the four subunits of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Changes in the spectrum of this group are produced by changes in pH, addition of DPN and DPNH, or addition of adenosine derivatives including adenosine diphosphoribose, ATP, AMP, and cyclic AMP. Below pH 7.8 the environment of the reporter group is apparently more polar than water while at higher pH there is a shift of the group to a less polar environment. This pH-dependent transition is consistent with the removal of a proton from a neighboring ionized residue with a pK of about 8.5 and is qualitatively similar to the effects observed with reporter-labeled chymotrypsin. Binding of DPN or DPNH also produces a shift of the reporter group to a nonpolar environment, although the changes are different for the two compounds. The alterations in Kdis, subunit interactions, and spectral properties of the reporter-labeled enzyme suggest that the perturbation of the reporter group is due to changes in conformation of the enzyme. The other adenosine compounds are also shown to bind to the enzyme and produce a shift in the spectrum of the reporter group. ATP is unique in that it is tightly bound to two sites on the enzyme but does not compete effectively with DPN. The properties of the reporter-labeled enzyme appear to correspond to those of the normal acyl enzyme intermediate and thus it provides a useful tool for studying the properties of this intermediate.