title: Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes.
abstract: 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)
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Benen
authors.forename: W
authors.initials: W
authors.lastname: van Berkel
authors.forename: N
authors.initials: N
authors.lastname: Dieteren
authors.forename: D
authors.initials: D
authors.lastname: Arscott
authors.forename: C
authors.initials: C
authors.lastname: Williams
authors.forename: C
authors.initials: C
authors.lastname: Veeger
authors.forename: A
authors.initials: A
authors.lastname: de Kok
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
chemical_list: D005971:Glutamates; D006639:Histidine; D011392:Proline; D008058:Dihydrolipoamide Dehydrogenase