Kinetic and mechanistic studies on the reduction of melilotate hydroxylase by reduced pyridine nucleotides.

L. M. Schopfer, V. Massey

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18 Scopus citations

Abstract

The reduction of the melilotate hydroxylase . 2-OH-phenyl propionate complex by NADH and reduced 3-acetyl pyridine adenine dinucleotide (AcPyNADH) has been investigated using steady state kinetic and rapid reaction techniques. Reduction by NADH appeared to involve only one charge-transfer-type intermediate (between reduced enzyme and NAD) as previously described (Strickland, S., and Massey, V. (1973) J. Biol. Chem. 248, 2953-2962). Reduction by AcPyNADH was shown to involve two charge-transfer-type intermediates. The first was between oxidized enzyme and AcPyNADH and the second was between reduced enzyme and AcPyNAD. Reaction of AcPyNADH with oxidized enzyme . 2-OH-phenyl propionate complex to form the first charge-transfer complex reached equilibrium within the mixing time of the stopped flow apparatus (5 ms). Subsequent steps in the reaction appeared to be first order and were independent of the AcPyNADH concentration. An 8-fold deuterium isotope effect on the step involving flavin reduction was found when reduced 3-acetyl[4A-2H]pyridine adenine dinucleotide (AcPyNADD) was used as the reductant. Analysis of the rapid reaction results for the reaction of oxidized pyridine nucleotide with reduced enzyme . 2-OH-phenyl propionate complex indicated the presence of two forms of reduced enzyme (in equilibrium) of which only one form was capable of reacting with the oxidized pyridine nucleotide. Based on the rapid reaction data, a mechanism for the reduction half-reaction is proposed. The turnover number calculated from this mechanism is in good agreement with that determined from the steady state data.

Original languageEnglish (US)
Pages (from-to)10634-10643
Number of pages10
JournalJournal of Biological Chemistry
Volume254
Issue number21
StatePublished - Nov 10 1979

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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