Synthesis of 4R- and 4S-tritium labeled NADPH for the determination of the coenzyme stereospecificity of NADPH: protochlorophyllide oxidoreductase.
A rapid and easy method for the production of both the 4R and 4S tritium labeled isomers of either NADH or NADPH has been developed. The method requires the use of only a single labeled compound (D-[1(-3)H] glucose), and two enzymes (glucose dehydrogenase from Bacillus sp. and alcohol dehydrogenase from Thermoanaerobium brockii) which are specific for the pro S and pro R hydrogens, respectively, of either NADH or NADPH. The 4R and 4S tritium labeled isomers of NADPH have been used to determine that NADPH:protochlorophyllide oxidoreductase from etiolated wheat was specific for the pro S hydrogen of NADPH.
Valera
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Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Fung
M
M
Wessler
A N
AN
Richards
W R
WR
D007553:Isotope Labeling / Q000379:methods; D009249:NADP / Q000138:chemical synthesis* / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010944:Plants / Q000201:enzymology; D011865:Radioisotope Dilution Technique; D013237:Stereoisomerism; D014908:Triticum / Q000201:enzymology; D014316:Tritium
D014316:Tritium; D009249:NADP; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase