Effects of Feedback Modifiers on Mutationally Altered Threonine Deaminases of Rhodopseudomonas spheroides

Abstract

Abstract Preliminary-kinetic analyses with two mutationally altered threonine deaminases, PR-TDI and PR-TDII, as well as the wild type enzyme of Rhodopseudomonas spheroides with l-threonine and l-serine as substrates reveal large differences in the substrate-binding abilities and significant variations in the homotropic interactions. The wild type enzyme shows sigmoid threonine saturation kinetics whereas the mutant enzymes reveal normal Michaelis-Menten behavior. The slope of the Hill plots with threonine as substrate indicates strong cooperative interactions for the wild type enzyme, while the slopes obtained for the mutant enzymes indicate lack of homotropic interactions. Similar experiments with l-serine as the substrate suggest loss of cooperativity with PR-TDI but not with PR-TDII. The substrate concentrations required for one-half maximal velocity, S0.5, with respect to both substrates are different for the mutationally altered enzymes as compared to the wild type enzyme. The dual effects of l-valine for activation and inhibition of enzyme activity are strongly dependent on the particular substrate used. With threonine as substrate, the activity of PR-TDI is stimulated at low levels of valine and inhibited at higher concentrations of this amino acid. The activity of PR-TDII is not stimulated by valine, but significant inhibition is observed at high valine concentrations. In contrast to the enzyme from the wild type organism, with serine as the substrate, no stimulation of enzyme activity by valine is observed with either PR-TDI or PR-TDII. Valine inhibits the activity of both mutant enzymes; with PR-TDII, 20-fold higher levels of valine are required for similar inhibition, and the maximal inhibition is only 50%. The inhibitory effect of isoleucine on enzyme activity is generally similar when either threonine or serine is used as substrate. PR-TDII is about 1000-fold less sensitive to isoleucine inhibition than PR-TDI. The data presented here support the model proposed earlier that there are two binding sites for valine per enzyme molecule and that one of these sites is closely associated with a threonine-binding site, whereas the second one is identical with or closely overlapping that of the isoleucine site. Furthermore, it is concluded from these studies that threonine and serine occupy separate sites on the enzyme molecule.