Mechanism of reduction of quinones by Trypanosoma congolense trypanothione reductase.

A number of quinones were analyzed as substrates for trypanothione reductase from Trypanosoma congolense, an enzyme responsible for the protection of trypanosomes against oxidative stress. Using NADPH as substrate, the maximal rate of the steady-state reaction at pH 7.5 was between 24 and 1.6 s-1 for 11 quinone substrates. The biomolecular steady-state rate constants for quinone reduction, V/Km, ranged from 240 to 1.9 x 10(5) M-1 s-1, and their logarithms exhibited a hyperbolic dependence on the one-electron-reduction potentials of the quinone substrate. The addition of NADP+ stimulated these rates, with V/Km values increasing with an increasing NADP+/NADPH ratio. The results of alkylation of the cysteine residue in the two-electron-reduced enzyme by iodoacetamide indicate that these residues are not primarily involved in the reduction of these quinones. Single-electron reduction of benzoquinone constitutes 40% of the total electron transfer from NADPH to quinone in the absence of NADP+, and increases to 80% at NADP+/NADPH ratios greater than 10. These steady-state results were confirmed in pre-steady-state rapid reaction experiments. The rate of reduced enzyme oxidation by 1,4-benzoquinone is approximately 100 times faster in the presence of NADP+ than in its absence. The reactivities of various pyridine nucleotide liganded forms of EH2 for quinone reduction are presumably affected by the electron density at FAD. We suggest that one-electron reduction of quinones occurs at a site distinct from the two active sites involved in hydride ion transfer and disulfide reduction.

Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-872] and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451----Ala, His450----Phe and His450----Tyr appear to be almost inactive in both directions. Enzyme His450----Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451----Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455----Asp more than Glu455----Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455----Asp and Glu455----Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)