KINETIC MECHANISMS OF POLYPHOSPHATE GLUCOKINASE FROM MYCOBACTERIUM-TUBERCULOSIS

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes th e phosphorylation of glucose using inorganic polyphosphate [poly(P)1] or ATP. The steady-state kinetic mechanisms of the poly(P)- and ATP-de pendent glucokinase reactions were investigated using initial velocity , product inhibition, and dead-end inhibition analyses. In the poly(P) -dependent reaction, the enzyme follows an Ordered Bi Bi sequential me chanism with poly(P) binding to the enzyme first and glucose 6-phospha te dissociating last. Polyphosphate is utilized nonprocessively with a preference for longer chains due to higher k(cat)/K-m values, The lac k of inhibition at high poly(P) concentrations suggests that binding o f poly(P) as a product is nor favorable, In the ATP-dependent glucokin ase reaction. the data are also consistent with an Ordered Bi Si seque ntial mechanism, with ATP binding to the enzyme first, and glucose 6-p hosphate leaving last. At high concentrations, ATP displays competitiv e substrate inhibition with respect to glucose, which is consistent wi th the formation of an enzyme ATP ATP nonproductive complex. The overa ll catalytic efficiencies (k(cat)/KiaKb) of the poly(P)- and ATP-depen dent reactions are similar to 10(11) M(-2) s(-1) and similar to 10(8) M(-2) s(-1), respectively. The higher catalytic efficiency, high value of the substrate specificity constant (k(cat)/K-a) approaching a diff usion-controlled limit, and the absence of substrate inhibition in th; poly(P)-dependent reaction suggest that poly(P), rather than ATP, is the major phosphate donor for poly(P)-glucokinase in M. tuberculosis.