Substrate selectivity and kinetics for gamma-hydroxybutyrate dehydrogenase from ralstonia eutropha

Abstract

The current report uses cloned iron (II)-dependent gamma-hydroxybutyrate dehydrogenase (GHB-DH; EC 1.1.1.61) originally from the bacterium Ralstonia eutropha to test 42 natural and synthetic compounds for substrate activity. GHB is a human drug of abuse recently accepted as a natural neurotransmitter. In order of descending efficacy, good substrates of GHB-DH are trans-4- hydroxycrotonate, gamma-hydroxybutyrate, 3-hydroxypropylsulfonate (synthetic), and (RS)-2-methyl-4-hydroxybutyrate (synthetic). They each contain a primary alcohol, a singly charged, negative functional group located 3 carbon atoms from the alcohol, and a conformation presumably similar to that of substantially fixed trans- 4-hydroxycrotonate. Ethanol at > 1% (v/v) also is a substrate. The GHB-like drug of abuse (RS)-4-hydroxypentanoate and other analogues, homologues, and metabolites of GHB are poor or non substrates. No other good natural substrate of GHB-DH is likely to exist. GHB and 3-hydroxypropylsulfonate exhibit initial-velocity kinetics diagnostic of random-order binding by the alcohol and NAD+ to the dehydrogenase. When testing otherwise normal human urine or blood under the prescribed conditions, GHB-DH from Ralstonia eutropha will detect only ingested GHB.