GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species.
GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH4), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a Km for GTP of 6.5 micromoles. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56 degrees C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH4 inhibited enzyme activity by 25% at a concentration of 100 micromoles. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a Ki of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO2- plus NO3-. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH4.
He
Aimin
A
Division of Medicinal and Natural Products Chemistry and Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.
Rosazza
John P N
JP
D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D003470:Culture Media; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007042:Hypoxanthines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009615:Nocardia / Q000201:enzymology* / Q000254:growth & development
D003470:Culture Media; D004791:Enzyme Inhibitors; D007042:Hypoxanthines; D001708:Biopterins; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase; C017202:2,4-diaminohypoxanthine; C003402:sapropterin