CHARACTERIZATION OF THE HYDROLYTIC ACTIVITY OF A POLYCLONAL CATALYTICANTIBODY PREPARATION BY PH-DEPENDENCE AND CHEMICAL MODIFICATION STUDIES - EVIDENCE FOR THE INVOLVEMENT OF TYR AND ARG SIDE-CHAINS AS HYDROGEN-BOND DONORS

The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydr oxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antib ody preparation elicited in a sheep by use of an analogous phosphate i mmunogen, were shown to adhere closely to the Michaelis-Menten equatio n, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is disc ussed briefly. Both the k(cat) and k(cat)/K-m values were shown to inc rease with increase in pH across a pK(a) of approx, 9. Group-selective chemical modification studies established that the side chains of tyr osine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, his tidine or cysteine residues. The combination of evidence from the kine tic and chemical modification studies and from studies on the pH-depen dence of binding suggests that catalysis involves assistance to the re action of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This comb ination of hydrogen-bond donors appears to be a feature common to a nu mber of other hydrolytic catalytic antibodies, High-pK(a) acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.