Use of the sulphite adduct of nicotinamide-adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase
- PMID: 30452
- PMCID: PMC1185865
- DOI: 10.1042/bj1730959
Use of the sulphite adduct of nicotinamide-adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase
Abstract
1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).
Similar articles
-
The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase.Biochem J. 1973 Apr;131(4):739-48. doi: 10.1042/bj1310739. Biochem J. 1973. PMID: 4352914 Free PMC article.
-
Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies.Biochemistry. 1986 Jan 14;25(1):227-36. doi: 10.1021/bi00349a032. Biochemistry. 1986. PMID: 3513825
-
Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase.Biochem J. 1978 Aug 1;173(2):597-605. doi: 10.1042/bj1730597. Biochem J. 1978. PMID: 29604 Free PMC article.
-
Synthesis and properties of some new NAD analogues.Top Curr Chem. 1974;52:209-33. doi: 10.1007/3-540-06873-2_18. Top Curr Chem. 1974. PMID: 4376282 Review. No abstract available.
-
Immobilized cofactors and multi-step enzyme-systems.Adv Exp Med Biol. 1974;42(0):187-9. doi: 10.1007/978-1-4684-6982-0_15. Adv Exp Med Biol. 1974. PMID: 4367326 Review. No abstract available.
Cited by
-
Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.Protein Sci. 1992 Jul;1(7):892-901. doi: 10.1002/pro.5560010707. Protein Sci. 1992. PMID: 1304374 Free PMC article.
-
Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite.Appl Environ Microbiol. 2004 Nov;70(11):6800-8. doi: 10.1128/AEM.70.11.6800-6808.2004. Appl Environ Microbiol. 2004. PMID: 15528547 Free PMC article.
-
Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.ACS Catal. 2023 Jan 20;13(2):1441-1448. doi: 10.1021/acscatal.2c05461. Epub 2023 Jan 10. ACS Catal. 2023. PMID: 37886035 Free PMC article.
-
Regeneration of nicotinamide cofactors for use in organic synthesis.Appl Biochem Biotechnol. 1987 Mar;14(2):147-97. doi: 10.1007/BF02798431. Appl Biochem Biotechnol. 1987. PMID: 3304160 Review.
-
Role of lipid composition and lipid peroxidation in the sensitivity of fungal plant pathogens to aluminum chloride and sodium metabisulfite.Appl Environ Microbiol. 2007 May;73(9):2820-4. doi: 10.1128/AEM.02849-06. Epub 2007 Mar 2. Appl Environ Microbiol. 2007. PMID: 17337539 Free PMC article.
References
MeSH terms
Substances
LinkOut - more resources
Full Text Sources