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

Abstract

GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, 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 pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin. The enzymes of this pathway are therefore potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate constant 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 by multiwavelength photometry are well in line with these data. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as substrate for GTP cyclohydrolase II but does not fulfill the criteria for a kinetically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under release of pyrophosphate. The covalently bound phosphoguanosyl moiety is proposed to undergo rapid hydrolytic release of formate from the imidazole ring and/or hydrolytic cleavage of the phosphodiester bond.