Mechanistic studies of choline oxidase with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde

被引:41
作者
Fan, F
Germann, MW
Gadda, G
机构
[1] Georgia State Univ, Dept Chem, Atlanta, GA 30302 USA
[2] Georgia State Univ, Dept Biol, Atlanta, GA 30302 USA
[3] Georgia State Univ, Ctr Biotechnol & Drug Design, Atlanta, GA 30302 USA
关键词
D O I
10.1021/bi0517537
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions in which betaine aldehyde is formed as an intermediate. The chemical mechanism for the oxidation of choline catalyzed by choline oxidase was recently elucidated by using kinetic isotope effects [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 2067-2074]. In this study, the oxidation of betaine aldehyde has been investigated by using spectroscopic and kinetic analyses with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde. The pH dependence of the k(cat)/K-m, and k(cat) values with betaine aldehyde showed that a catalytic base with a pK(a) of similar to 6.7 is required for betaine aldehyde oxidation. Complete reduction of the enzyme-bound flavin was observed in a stopped-flow spectrophotometer upon anaerobic mixing with betaine aldehyde or choline at pH 8, with similar k(red) values >= 48 s(-1). In contrast, only 10-26% of the enzyme-bound flavin was reduced by 3,3-dimethylbutyraldehyde between pH 6 and 10. Furthermore, this compound acted as a competitive inhibitor versus choline. NMR spectroscopic analyses indicated that betaine aldehyde exists predominantly (99%) as a diol form in aqueous solution. In contrast, the thermodynamic equilibrium for 3,3-dimethylbutyraldehyde favors the aldchyde (>= 65%) over the hydrated form in the pH range from 6 to 10. The keto species of 3,3-dimethylbutyraldehyde is reactive toward enzymic nucleophiles, as suggested by the kinetic data with NAD(+)-dependent yeast aldehyde dehydrogenase. The data presented suggest that choline oxidase utilizes the hydrated species of the aldehyde as substrate in a mechanism for aldehyde oxidation in which hydride transfer is triggered by an active site base.
引用
收藏
页码:1979 / 1986
页数:8
相关论文
共 52 条
[31]   ALDEHYDE DEHYDROGENASES AND THEIR ROLE IN CARCINOGENESIS [J].
LINDAHL, R .
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1992, 27 (4-5) :283-335
[32]   Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans [J].
Marchal, S ;
Cobessi, D ;
Rahuel-Clermont, S ;
Tête-Favier, F ;
Aubry, A ;
Branlant, G .
CHEMICO-BIOLOGICAL INTERACTIONS, 2001, 130 (1-3) :15-28
[33]   Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans [J].
Marchal, S ;
Branlant, G .
BIOCHEMISTRY, 1999, 38 (39) :12950-12958
[34]   Ligand-induced conformational changes of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves affecting the reactivity of the catalytic thiol [J].
Muñoz-Clares, RA ;
González-Segura, L ;
Mújica-Jiménez, C ;
Contreras-Díaz, L .
CHEMICO-BIOLOGICAL INTERACTIONS, 2003, 143 :129-137
[35]   PURIFICATION AND CHARACTERIZATION OF HISTIDINOL DEHYDROGENASE FROM CABBAGE [J].
NAGAI, A ;
SCHEIDEGGER, A .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1991, 284 (01) :127-132
[36]   STRUCTURAL AND FUNCTIONAL CONSERVATION OF HISTIDINOL DEHYDROGENASE BETWEEN PLANTS AND MICROBES [J].
NAGAI, A ;
WARD, E ;
BECK, J ;
TADA, S ;
CHANG, JY ;
SCHEIDEGGER, A ;
RYALS, J .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (10) :4133-4137
[37]   The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition [J].
Okamoto, K ;
Matsumoto, K ;
Hille, R ;
Eger, BT ;
Pai, EF ;
Nishino, T .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (21) :7931-7936
[38]   Metabolism of isovanillin by aldehyde oxidase, xanthine oxidase, aldehyde dehydrogenase and liver slices [J].
Panoutsopoulos, GI ;
Beedham, C .
PHARMACOLOGY, 2005, 73 (04) :199-208
[39]   Contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase on the oxidation of aromatic aldehydes [J].
Panoutsopoulos, GI ;
Kouretas, D ;
Beedham, C .
CHEMICAL RESEARCH IN TOXICOLOGY, 2004, 17 (10) :1368-1376
[40]  
Peddie BA, 1998, FEMS MICROBIOL LETT, V160, P25