Donald L. Sloan

A high-pressure liquid chromatography procedure for monitoring nicotinate phosphoribosyltransferase activity

Abstract A high-pressure liquid chromatography (hplc) procedure has been designed to monitor the reaction catalyzed by nicotinate phosphoribosyltransferase (N-PRTase). Two substrates (ATP and nicotinate) and two products of this reaction (nicotinate mononucleotide and ADP) can be resolved at pH 8 on a Waters μBondapak C18 column, and the concentration of each reactant in the isocratic eluant (25 m m (NH4)3PO4) can be determined spectroscopically at 254 nm. This separation method allows an analysis of the disappearance of substrates and the appearance of products simultaneously during the time course of the enzyme-catalyzed reaction. We have employed this new N-PRTase assay system: (1) to prove that a 1:1 stoichiometry exists between the enzymatic ATP hydrolysis and nicotinate mononucleotide formation and (2) to reveal for the first time an inhibition of the overall reaction by phosphoribosyl α-1-pyrophosphate at relatively low ATP concentrations.

Activation of hypoxanthine/guanine phosphoribosyltransferase from yeast by divalent zinc and nickel ions.

We have observed previously that the reactions catalyzed by hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) are activated by Mg(II), Mn(II), and Co(II), and we have defined the mechanism by which these activations proceed [Biochemistry 22, 3419-3424 (1983)]. A more extensive survey of the kinds of metal ions that will activate the HGPRTase catalysis now has been completed through the use of an HPLC assay procedure. Although Fe(II) and Ca(II) are unable to activate this reaction, a significant activation was achieved with the addition of spectroscopically pure Zn(II) to the assay solution. In addition some IMP synthesis resulted from the addition of Ni(II) to the assay mixture. Both the Zn(II) and Ni(II) kinetic effects on HGPRTase over a limited metal ion concentration range have been analyzed through the use of curve-fitting exercises. These results, in addition to the similar pH profiles for the activations by Mg(II), Mn(II), Co(II), and Zn(II), suggest that all of these metal ions activate the HGPRTase-catalyzed synthesis of IMP by way of the same mechanism [model II as defined by London and Steck, Biochemistry 8, 1767-1779 (1969)], during which two divalent ions bind to the HGPRTase active site per molecule of PRibPP.

Raman spectroscopy of liver alcohol dehydrogenase

We report the Raman spectrum of liver alcohol dehydrogenase in solution. The enzymes secondary structure as determined from an examination of the Raman bands is slightly different than that found in crystals by X-ray diffraction.

Enzymatic assay procedures that employ high-performance liquid chromatography: Competition between phosphoribosyltransferases for a common substrate

A survey of the phosphoribosyltransferase (PRTase) activities in yeast has been accomplished using reversed-phase high-performance liquid chromatographic assay procedures. The following bases were observed to be utilized during phosphoribosyl pyrophosphate (PRibPP)-dependent nucleotide syntheses: adenine, xanthine, hypoxanthine, guanine, uracil, orotate, nicotinamide, nicotinate and quinolinate. Gradient elution procedures have also been perfected that allow the separation of the two following sets of PRTase assay components: (1) adenosine monophosphate, nicotinate mononucleotide, orotate, adenosine triphosphate, nicotinate, adenosine diphosphate, inosine monophosphate and hypoxanthine, and (2) nicotinate mononucleotide, nicotinamide mononucleotide, adenosine triphosphate, nicotinate, adenosine diphosphate and nicotinamide. Separation 1 has been employed to examine the PRibPP allocation among the hypoxanthine PRTase, orotate PRTase and nicotinate PRTase catalyzed reactions, whereas separation 2 has been employed to define the role that ATP plays in the nicotinamide PRTase-catalyzed reaction along with the allocation of nicotinamide between the reactions catalyzed by nicotinamide PRTase and nicotinamide deamidase.

Orotate phosphoribosyltransferase from yeast: Studies of the structure of the pyrimidine substrate binding site☆

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).

Enzymatic kinetic analyses that emplo high-performance liquid chromatography : Competition betwen orotate- and hypoxanthine/guanine-phosphoribosyltransferases for a common substrate

Abstract Enzymatic assay procedure that employ high-performance liquid chromatography (HPLC) have been proven to be sensitive and versatile methods for accomplishing kinetic analyses of enzyme-catalyzed reactions, with nucleotides as substrates or products. Both orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) have been purified from Bakers yeast and analyzed kineticaly using a modification of published HPLC procedures. Because these two enzymes exist in the cytosol of yeast and might compete for the limiting ( ≈15 μ M ) concentration of phosphoribosyl α-1-pyrophosphate (PRibPP), we elected to examine both equilibrium and steady-state effects of one enzymatic reaction on the other with HPLC. First, under the condition of equivalent mass concentrations of OPRTase and HGPRTase, the initial rate of orotidine monophosphate synthesis and the equilibrium state were greatly affected by the presence of HGPRTase acitivity. In conrast, the presence of the OPRTase activity had no effect on the HGPRTase-catalyzed reaction under these conditions. Second, to examine a competition by these enzymes for PRibPP in vivo , we have established that the total activities (units/ml) of OPRTase and HGPRTase in yeast cell extracts were f740 units/ml and 450 units/ml, respectively )a 1.7:1 ratio). These relative activities were then employed in an in vitro reaction competition analysis. The results were similar to the those obtained from experiments where equivalent OPRTase and HGPRTase activities were employed and reveal profound initial velocity and equilibrium effecfs one one reaction on the other. Thus a real competition between these enzymes for PRibPP may occur in the yeast cell cytosol, as determined by this unique HPLC competition assay procedure.

Studies of the Catalytically-Active Form of Hypoxanthine-Guanine Phosphoribosyltransferase from Yeast

Studies of the kinetic (1) and structural (2) properties of yeast hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase), an enzyme first purified by Schmidt et al. (3), have been completed recently. This HG-PRTase is composed of two polypeptide subunits(26,000 m.w.), as determined by SDS-gel electrophoresis (2) and catalyzes the synthesis of either IMP or GMP through the use of an ordered Bi Bi kinetic mechanism (1). However, since electro-phoretic studies are conducted at concentrations much higher than those employed during kinetic analyses, the observed dimeric HG-PRTase, may not necessarily be the form of the enzyme responsible for catalysis. In this paper we describe results which suggest the existence of a catalytically-active monomeric HG-PRTase which functions as an enzyme-metal ion complex.

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: Kinetic analysis with chromium(III) pyrophosphate

The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.

Enzymatic coupled assay procedures that employ high-performance liquid chromatography: The synthesis of orotidylate from ribose

High-performance liquid chromatographic assay procedures have been designed to monitor the catalytic activities of ribokinase and phosphoribosyl alpha-1-pyrophosphate (PRibPP) synthetase. These methods are only of qualitative value, when crude protein extracts are to be examined, because of the presence of myokinase. However, the product of the PRibPP synthetase reaction, can be detected quantitatively even in crude protein extracts through the addition of two enzymes (orotate phosphoribosyltransferase and inorganic pyrophosphatase) that catalyze the conversion of PRibPP into a spectroscopically detectable nucleotide product (orotidylate).

Determination of a Histidine Residue at the Yeast Orotate Phosphoribosyltransferase Active-Site

A purified preparation of orotate phosphoribosyltransferase(0-PRTase) from yeast has been shown to catalyze the formation of orotidine monophosphate (OMP) from orotate and 5-phosphoribosyl α-1-pyrophosphate (PRibPP) through the use of a ping pong kinetic mechanism (1) in the presence of an optimum concentration of Mgll(2). The present investigation was initiated to search for nucleo-philic active-site residues of 0-PRTase which would stabilize (in an SN1 elimination) or react with (in a triple SN2 displacement) the enzyme-phosphoribosyl intermediate, formed as a consequence of this mechanism. Analysis of the amino acid composition, pH dependency of the 0-PRTase activity in the forward and reverse directions, and chemical modification of 0-PRTase using histidine-specific reagents have been accomplished.