GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species

Abstract

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.

FIG. 1.

Pathways for BH₄ biosynthesis and activity assay for GTPCH. PTPS, 6R-pyruvoyltetrahydropterin.

FIG. 2.

Elution profile of Nocardia GTPCH obtained by Ultragel AcA 34 column chromatography. ○, absorbance at 280 nm; ▪, enzyme activity.

FIG. 3.

SDS-PAGE of samples from the purification of Nocardia GTPCH. Lane 1, crude extract; lane 2, dialyzed ammonium sulfate precipitate; lane 3, pooled fractions from GTP-agarose; lane 4, pooled fractions from DEAE Sepharose; lane 5, pooled fractions from Ultragel AcA 34; lane 6, control with sample buffer; lane 7, molecular mass markers (phosphorylase b [97.4 kDa], serum albumin [66.2 kDa], ovalbumin [45 kDa], carbonic anhydrase [31 kDa], trypsin inhibitor [21.5 kDa], and lysozyme [14.4 kDa]).

FIG. 4.

Effects of DAHP and LNMA on NO₂⁻ plus NO₃⁻ formation in Nocardia. Controls were cells grown in phenylalanine-free medium (lane 1). Cells grown in Phe-containing medium (lane 2) were incubated with various concentrations of DAHP or LNMA for 24 h. The resulting supernatants were assayed for NO₂⁻ plus NO₃⁻, as described in Materials and Methods. Data represent means ± standard deviations (n = 3). *, P < 0.05 versus the respective value obtained from cells without DAHP or LNMA treatment.