1999年1月8日
Purification and characterization of NAD:penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation
Journal of Biological Chemistry
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- 巻
- 274
- 号
- 2
- 開始ページ
- 795
- 終了ページ
- 800
- 記述言語
- 英語
- 掲載種別
- DOI
- 10.1074/jbc.274.2.795
A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (K(m) = 13.0 mM) and L-penicillamine (K(m) = 6.5 mM)
other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.
other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.
- ID情報
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- DOI : 10.1074/jbc.274.2.795
- ISSN : 0021-9258
- PubMed ID : 9873018
- SCOPUS ID : 0033534616