INITIAL RATE AND EQUILIBRIUM ISOTOPE-EXCHANGE STUDIES ON THE ATP-DEPENDENT ACTIVITY OF POLYPHOSPHATE GLUCOKINASE FROM PROPIONIBACTERIUM-SHERMANII

Polyphosphate glucokinase [EC 2.7.1.63] catalyzes the phosphorylation of glucose using either inorganic polyphosphate [poly(P)] or ATP as th e phosphoryl donor. Both activities purified from Propionibacterium sh ermanii are the functional properties of a single enzyme with separate binding sites for the two phosphoryl donor substrates. The enzyme was found to utilize poly(P) much more efficiently than it does ATP, with a k(cat)/K-poly(P) to k(cat)/K-ATP Patio of 2800. The catalytic const ant for poly(P) is about 2-fold higher than for ATP. Other nucleotides like GTP and dATP also served as substrates with good efficiencies. T he ATP-dependent reaction was analyzed using steady-state kinetics and isotopic exchange kinetics at chemical equilibrium. Intersecting init ial velocity patterns for both glucose and ATP indicate sequential add ition of substrates. Product inhibition studies resulted in two compet itive and two noncompetitive patterns, which is characteristic of a Th eorell-Chance mechanism or a random mechanism with two dead-end comple xes. Results of isotope exchange experiments, however, rule out a Theo rell-Chance mechanism, as well as a truly random mechanism, They are m ost consistent with a partially random mechanism (although a kinetical ly compulsory order of substrate binding is not excluded), where gluco se is preferentially bound to free enzyme before ATP, and ADP is prefe rentially released as the first product, followed by glucose 6-phospha te. Dead-end inhibition analysis confirms this order of substrate bind ing. Competitive inhibition of ADP vs ATP is explained as resulting pr imarily from binding as a dead-end inhibitor (E . Glc . ADP) and not a s a product. Another weaker abortive complex, E . ATP . GGP, is also f ormed. The chemical transformation or the release of ADP is the rate-l imiting step in ATP utilization.