KINETIC AND MODELING STUDIES OF NAD(-BOUND NAD(+) IN HORSE LIVER ALCOHOL-DEHYDROGENASE() AND POLY(ETHYLENE GLYCOL))

Poly(ethylene glycol)-bound nicotinamide adenine dinucleotide (PEG-NAD (+)) has been successfully employed in the continuous production of L- amino acids from the corresponding alpha-keto acids by stereospecific reductive amination. Like many other dehydrogenases also horse liver a lcohol dehydrogenase (HLADH) appears to be active with PEG-NAD(+) as c oenzyme, although the turnover number is three to four times lower. Th e possibilities were considered that the PEG-tail of a PEG-NAD(+) boun d to one active site of the HLADH dimer prevents the binding of anothe r PEG-NAD(+) to the second site, or that the PEG-tail causes destabili zation of the active dimer. Both could be ruled out by kinetic studies . Neither can the observed lower intrinsic reactivity of PEG-NAD(+) ac count for the diminished activity of the enzyme. Molecular dynamics st udies, on the other hand, show that the pulling action of the polymer chain shifts the NAD position in the active site in the outside direct ion, causing small but significant changes in the enzyme/coenzyme inte ractions of a sufficient extent to explain the experimental results.