Abstract:
The kinetic study of the coupled enzymatic reaction involving monomeric yeast
hexokinase PII (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDH) yields a Michaelis
constant of 0.15 ± 0.01 mM for D-glucose. At pH 8.7 HK is present in monomeric form. The
addition of polyethylene glycol (PEG), to the reaction mixture increased the affinity of HK for
glucose, independent ofMW of the PEG from 2000 to 10000. The osmotic stress exerted by PEG
can be used to measure the change in number of water molecules that accompany enzyme
conformational changes (Rand, et al., 1993). Results indicate that the G-6-PDH is not osmotically
sensitive and thus, the change in the number of PEG-inaccessible water molecules (ANw)
measured in the coupled reaction is only the difference between the glucose-bound and glucosefree
conformations of HK. ANw ~ 450 with PEGs of MW > 2000 under conditions for both
binding (Reid and Rand, 1997) and kinetic assays. The contribution water may play in the binding
of ATP (Km = 0.24 + 0.02 mM) has also been examined. It was found that in this case ANw =
(for osmotic pressures < 2.8x10* dynes/cm^), suggesting no additional numbers of waters are
displaced when ATP binds to HK. Osmotic pressure experiments were also performed with
dimeric HK. It was determined that both the monomeric and dimeric forms of HK give the same
ANw under low pressures. If this large ANw is due to conformational flexibility, it would appear
that the flexibility is not reduced upon dimerization ofthe enzyme.
