Purification, kinetic mechanism and regulation of nitrate reductase from candida utilis

Abstract

Nitrate is a major source of inorganic nitrogen utilized by most plants, some algae, fungi, and a variety of bacteria. This fundamental biological process of nitrate assimilation in most systems involves the reduction of the highly oxidized form of inorganic nitrogen to ammonia. This is mediated by two metallo-proteins, namely, nitrate reductase and nitrite reductase. These enzymes catalyze the stepwise reduction of nitrate to nitrite and nitrite to ammonia, respectively.

NO 3

Nitrate reductase NO 2

Nitrite reductase NH 4 + NO 3

Nitrate reductase

NO 2

Nitrite reductase

NH 4 +

Nitrate reductase, the first enzyme in the assimilatory pathway, is thought to play a critical role in the regulation of the pathway. Hence, it has drawn the attention of researchers. Enzymology and genetic analysis of this enzyme have been carried out in different systems. Among fungi, Aspergillus and Neurospora have been extensively used in these studies. However, detailed knowledge of this enzyme in yeast has been limited to a few species, and many gaps exist in our understanding of the kinetic mechanisms reported for this enzyme from different systems. It is reported to exhibit either a rapid random equilibrium or a ping-pong mechanism. Despite numerous correlations of various environmental factors to nitrate reductase activity, the levels at which regulation takes place are far from clear. In the first chapter, a brief survey of literature on various aspects of nitrate reductase is presented.

The assimilatory nitrate reductase, NAD(P)H-nitrate oxidoreductase (EC 1.6.6.2) from nitrate-utilizing yeast Candida utilis, was isolated and purified to electrophoretic homogeneity. The purification protocol, involving a combination of conventional purification methods and affinity chromatography using Blue Sepharose, proved to be advantageous and gave better results than previously reported in this system. The enzyme is purified by over 600-fold with a yield of 60%. The enzyme is a dimer with a molecular mass of 200,000 ± 3,000 daltons. It is composed of two identical subunits of molecular mass 100,000 daltons. In subunit structure and molecular weight, it resembles enzymes isolated from other sources. The optimal pH, temperature, and time course for maximum activity of the purified enzyme have been established. The affinity of the enzyme for the substrates nitrate (125 M) and NADH (45 M) has been determined. The enzyme has a pI of 6.5. Spectral analysis of the purified enzyme indicated the presence of cytochrome b 57 57

.

The enzyme is inhibited by sulphydryl reagents, indicating that sulphydryl groups are involved at the active center of the enzyme or are required for enzyme activity. The inhibition by N-ethylmaleimide was prevented by glutathione, which further confirmed the above finding. The inhibition by metal-binding agents like cyanide and thiourea is dependent on the oxidation–reduction state of the enzyme. They exhibited a non-competitive type of inhibition on the enzyme when pre-incubated in the presence of NADH. Cyanide and thiourea failed to inhibit nitrate reductase in the presence of ferricyanide.

In the third chapter, the kinetic properties of the enzyme have been investigated. Results from initial velocity studies for bisubstrate enzymes allow classification of the kinetic mechanism into sequential or ping-pong. Inhibition patterns obtained with the products at subsaturating and saturating concentrations of the fixed substrate further help in distinguishing and confirming a particular mechanism. The kinetic analysis of the purified enzyme from Candida utilis was carried out as described by Cleland. The analysis of the results indicates that the mechanism is an ordered sequential one. NAD(P)H binds to the enzyme prior to the addition of nitrate. A ternary complex is formed. The products are formed and released in the order: nitrite followed by NAD + + . Studies with the dead-end inhibitor thiocyanate also supported this mechanism.

In vivo regulation of nitrate reductase levels was examined in the presence of nitrate and ammonium. Cells grown in the presence of ammonium, when transferred to nitrate medium, showed nitrate reductase activity within 30 min, and the level reached a maximum by 3 hours. Nitrate reductase activity was absent both in ammonia-grown cells and in cells starved of nitrogen sources. When induced cells were transferred to ammonium medium, 50% loss in activity occurred in one and a half hours. Ouchterlony double diffusion tests revealed the absence of protein reacting with nitrate reductase antibody in ammonia-grown cells.

The synthesis of nitrate reductase protein, immunoprecipitable by its specific antibody, was examined by SDS-polyacrylamide gel electrophoresis of cell-free extracts pulse-labeled with 3 5 3 5S-methionine. The results revealed that while nitrate induced enzyme synthesis, ammonia completely abolished it. Quantitation of nitrate reductase protein by ELISA indicated that de novo synthesis of the enzyme occurs upon induction in the presence of nitrate. Ammonia prevents this induced synthesis. The absence of nitrate reductase protein in ammonia-grown cells was further confirmed by demonstrating the inability of extracts from ammonia-grown cells to relieve the inhibition of nitrate reductase activity by its specific antibody.

Using antibodies raised against purified nitrate reductase, polysomes enriched with nitrate reductase mRNA were selected. cDNA prepared from this message was used to screen a cDNA library prepared from poly(A) RNA from nitrate-grown cells. A nitrate reductase-specific clone, designated as pNAR6, was identified. Using this cDNA clone, steady-state nitrate reductase mRNA levels were measured in cells grown in nitrate and ammonium. Dot blot analysis revealed a 10-fold higher level of nitrate reductase mRNA in nitrate-grown cells. Therefore, nitrate regulates nitrate reductase at the transcriptional level. Regulation by ammonia occurs both at the transcriptional level (10-fold reduction in mRNA synthesis) as well as post-transcriptional and/or translational levels, since the mRNA made is not translated, as revealed by the absence of nitrate reductase protein in ammonia-grown cells.