Biosynthesis of riboflavin - Single turnover kinetic analysis of GTP cyclohydrolase II

GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5 -diamino-6-ribosylamino-4(3H)- Pyrimidinone 5'-phosphate (Compound 2), form ate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committed intermediate in the biosynthesis of the vitamin, riboflavin. Numerous path ogenic microorganisms are absolutely dependent on endogenous synthesis of r iboflavin. The enzymes of this pathway are therefore potential drug targets , and mechanistic studies appear relevant for development of bactericidal i nhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reac tion sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate cons tant of 0.064 s(-1), and the reaction product, Compound 2, is formed at an apparent rate constant of 0.062 s(-1). Stopped flow experiments monitored b y multiwavelength photometry are well in line with these data. 2-Amino-5-fo rmylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as subst rate for GTP cyclohydrolase II but does not fulfill the criteria for a kine tically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under rele ase of pyrophosphate. The covalently bound phosphoguanosyl moiety is propos ed to undergo rapid hydrolytic release of formate from the imidazole ring a nd/or hydrolytic cleavage of the phosphodiester bond.