Involvement of tryptophan(s) at the active site of polyphosphate/ATP glucokinase from Mycobacterium tuberculosis.

The glucokinase (EC 2.7.1.63) 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 sites was investigated by using N-bromosuccinimide (NBS) as a probe for the involvement of tryptophan in substrate binding and/or catalysis. NBS oxidation of the tryptophan(s) resulted in fluorescence quenching with concomitant loss of both the poly(P)- and ATP-dependent glucokinase activities. The inactivation by NBS was not due to extensive structural changes, as evidenced by similar circular dichroism spectra and fluorescence emission maxima for the native and NBS-inactivated enzyme. Both phosphoryl donor substrates in the presence of xylose afforded approximately 65% protection against inactivation by NBS. The Km values 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 tryptophan residue(s) were partially shielded by the substrates against quenching. The Stern-Volmer quenching constant (KSV) of the tryptophans in unliganded glucokinase was 3.55 M-1, while KSV 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 xylose+poly(P)5 and xylose+ATP against oxidation by NBS. The two protected peptides were determined to be identical by N-terminal sequence analysis and amino acid composition.(ABSTRACT TRUNCATED AT 250 WORDS)