Functional properties of the active core of human cystathionine beta-synthase crystals

Human cystathionine beta -synthase is a pyridoxal 5'-phosphate enzyme conta ining a heme binding domain and an S-adenosyl-L-methionine regulatory site. We have investigated by single crystal microspectrophotometry the function al properties of a mutant lacking the S-adenosyhmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are r eversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, whi ch takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate- dependent protein. The exposure of these crystals to increasing concentrati ons of the natural substrate L-serine readily led to the formation of the k ey catalytic intermediate alpha -aminoacrylate. The dissociation constant o f L-serine was found to be 6 mM, close to that determined in solution. The amount of the alpha -aminoacrylate Schiff base formed in the presence of L- serine was pH independent between 6 and 9, However, the rate of the disappe arance of the alpha -aminoacrylate, likely forming pyruvate and ammonia, wa s found to increase at pH values higher than 8, Finally, in the presence of homocysteine the alpha -aminoacrylate-enzyme absorption band readily disap pears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta -synthase crystals catalyze b oth beta -elimination and beta -replacement reactions. Taken together, thes e findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta -synthase.