Affinity labeling of succinyl‐CoA synthetase from Escherichia coli by the 2′,3′‐dialdehyde derivative of adenosine 5′‐diphosphate

Abstract

The 2',3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase. Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol ufl or, extrapolated, 2 mol/mol inactive a2& molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate t* succinyl-CoA exchange reaction, in comparison to the CoA tt succinyl-CoA exchange reaction. Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.Succinyl-CoA synthetase catalyzes the reaction shown in Eqn (1)( 1 1 where NDP and NTP represent purine ribonucleoside diphosphate and triphosphate, respectively. Partial reactions that reflect the catalytic steps of the synthetase activity are given in Eqns (2a -c).The enzymes from pig heart and rat liver have an aj?quaternary structure [I, 21, whereas the Escherichia coli enzyme can be described as an u& protein [3]. The u subunit is phosphorylated during the reaction [1,3]. The subunit contains an important sulfhydryl group that is labeled by CoA thiosulfonate, an affinity labeling agent of the enzyme [4,5]. These data are consistent with the view that the active site of the enzyme is located at the point of contact between subunits. Recently, it has been hypothesized that E. coli succinylCoA synthetase exhibits alternating sites cooperativity [6]. In the model, the two active sites are viewed as being alternatively productive with substrate (ATP or succinyl-CoA) binding at the nonproductive site bringing about release of product (succinvl-CoA or ATP) from the oroductive site. The phenomena associated with the alternating sites concept were not exhibited by the up pig heart enzyme. Other data that are consistent with the hypothesis have also been publishedWe have studied the NTP(NDP) binding site of the UP rat liver enzyme with oGDP as the probe [2]. This compound brough about dephosphorylation of the enzyme and subsequent rapid inactivation of the enzyme. The dephosphorylated enzyme was also much more rapidly inactivated by oGDP than the phosphorylated enzyme. Data indicative of binding to both u and p subunits was obtained, suggesting participation of both polypeptide chains in the make-up of the NTP(NDP) binding site. It was thus logical to extend the study to the E. coli enzyme with its two active sites that appear to interact. In the present study, the effects of oADP have been examined as an affinity label of the E. coli enzyme.

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

“…It appears, then, that while 3-subunit that is modified with respect to one tryptophan residue is catalytically inactive, it is nevertheless able to refold to the extent required for combination with refolded -subunit to yield a form of the enzyme that is capable of normal interaction with ATP. It is also conceivable that the /3-subunit contributes to the ATP binding domain (Nishimura et al, 1983). By the criterion of gel filtration (Figure 5), refolded enzyme containing modified /3-subunit appears to be of normal molecular size.…”