INVOLVEMENT OF TRYPTOPHAN(S) AT THE ACTIVE-SITE OF POLYPHOSPHATE ATP GLUCOKINASE FROM MYCOBACTERIUM-TUBERCULOSIS

The glucokinase (EC2.7.1.63) from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphate (poly(P) ) or ATP as the phosphoryl donor. The nature of the poly(P) and ATP si tes was investigated by using N-bromosuccinimide (NBS) as a probe for the involvement of tryptophan in substrate binding and/or catalysis. N BS oxidation of the tryptophan(s) resulted in fluorescence quenching w ith concomitant loss of both the poly(P)- and ATP-dependent glucokinas e activities. The inactivation by NBS was not due to extensive structu ral changes, as evidenced by similar circular dichroism spectra and fl uorescence emission maxima for the native and NBS-inactivated enzyme. Both phosphoryl donor substrates in the presence of xylose afforded ap proximately 65% protection against inactivation by NBS. The K(m) value s of poly(P) and ATP were not altered due to the modification by NBS, while the catalytic efficiency of the enzyme was decreased, suggesting that the essential tryptophan(s) are involved in the catalysis of the substrates. Acrylamide quenching studies indicated that the tryptopha n residue(s) were partially shielded by the substrates against quenchi ng. The Stern-Volmer quenching constant (K(SV)) of the tryptophans in unliganded glucokinase was 3.55 M-1, while K(SV) values of 2.48 and 2. 57 M-1 were obtained in the presence of xylose+poly(P)5 and xylose+ATP , respectively. When the tryptophan-containing peptides were analyzed by peptide mapping, the same peptide was found to be protected by xylo se+poly(P)5 and xylose+ATP against oxidation by NBS. The two protected peptides were determined to be identical by N-terminal sequence analy sis and amino acid composition. It is proposed from these results that one or both of the tryptophans present in the protected peptide may b e located at a common catalytic center and that this peptide may const itute part of the poly(P) and ATP binding regions.