Ornithine Transcarbamylase from Streptococcus faecalis and Bovine Liver: III. EFFECTS OF CHEMICAL MODIFICATIONS OF SPECIFIC RESIDUES ON LIGAND BINDING AND ENZYMATIC ACTIVITY

Abstract

Abstract Ornithine transcarbamylase from Streptococcus faecalis has two sulfhydryl groups per monomer that react with 5,5'-dithiobis (2-nitrobenzoate) (DTNB) in urea: one of these reacts rapidly with p-hydroxymercuribenzoate (p-HMB) in glycylglycine buffer (pH 7.8). The enzyme from bovine liver has slightly less than three per monomer that react with DTNB in urea: two of these react with p-HMB at pH 7.8 and one reacts rapidly with 0.25 mm DTNB at pH 7.8 without urea and in the presence of carbamyl-P plus norvaline. The sulfhydryl groups in this last category reacted without loss of activity with all of the reagents that were tried. Both enzymes were inactive in the presence of an amount of p-HMB stoichiometric with the reactive sulfhydryl groups. The addition of EDTA slowed down the inactivation by p-HMB to a measurable rate, which was decreased in the presence of carbamyl-P, phosphate, and more effectively, phosphate plus ornithine. These sulfhydryl groups reacted with iodoacetamide only under conditions that are known to cause a conformational change, exposing this group, i.e. with the inactive dimer of the S. faecalis enzyme and in 1 m KCl, pH 10, 0° for the bovine enzyme. They reacted relatively rapidly with cystamine at pH 8.5 in a pseudo-first order reaction that led to complete inactivation. The binding of carbamyl-P was not affected, but the derivatives did not bind norvaline. In the presence of carbamyl-P, the inactivation was still a first order reaction, but the rate decreased. The dissociation constants for carbamyl-P calculated from the change in the rate with concentration agreed with those determined from direct measurement of the binding. The reaction with dithiodiglycol was less rapid than with cystamine, and the derivative of the bovine enzyme with less than 2% of the essential sulfhydryl groups remaining had about one-half the activity of the native enzyme at pH 8.5. The concentration of the substrates, particularly ornithine, required to saturate it were, however, much higher, as were the dissociation constants for carbamyl-P and norvaline. The S. faecalis enzyme lost activity on incubation with acetylimidazole, 50 mm at 25° in 0.1 m sodium morpholinoethanesulfonate (pH 6.0) for 20 min. Neither the sulfhydryl groups nor tyrosine residues had reacted, but 8.3 amino groups per monomer were acetylated. The inactivation was less rapid in the presence of carbamyl-P plus norvaline, and 1.3 fewer amino groups had reacted. Reaction of a single amino group per monomer of the S. faecalis enzyme with 2,4,6-trinitrobenzenesulfonate (TNBS) at pH 6.0, 25°, caused complete inactivation; no reaction occurred in the presence of carbamyl-P. The specificity is due to the reagent forming a complex with the enzyme at the site of the reaction. From the pH dependence of its dissociation constant, the binding was found to depend on the protonation of a group on the enzyme with pK 6.1. The pH independent value of Ktnbs is 5.4 mm. A number of competitive inhibitors of TNBS were found; the most effective were anions with a bulky nonpolar side chain. Both enzymes, their essential sulfhydryl groups having been protected by oxidation to the aminoethylthio mixed disulfides, reacted with iodoacetate, 10 to 50 mm at pH 5.0 to 5.5, 30–40°, with loss of activity. Carboxymethyl groups (1.4) per monomer were incorporated from [1-14C]iodoacetate when 90% of the activity was lost. The radioactive components separated from the acid hydrolysates of the alkylated derivatives by chromatography in the amino acid analyzer appeared in the same positions as the components separated from the hydrolysate of methionine [14C]carboxymethylsulfonium iodide. The alkylation of methionine did not affect the binding of carbamyl-P, but norvaline was not bound to the derivative.