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.     

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.     

The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation

We have developed a method of relating changes in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) activity to the rate of phosphoribosyl pyrophosphate (PRPP) synthesis in isolated cell lines and in co-cultures of different cell lines. Using this approach, we have determined the response of the HGPRTase activity of communication-competent and communication-incompetent cells to changes in PRPP content. The HGPRTase activity of HGPRT+ communication-competent NS cells responds to changes of their own PRPP level, as well as to changes of the PRPP level of HGPRT- cells with which they are co-cultured. In contrast, the HGPRTase activity of the HGPRT+, but communication-incompetent L929 cells responds to changes of their own PRPP content but not to changes of the PRPP content of the cocultured HGPRT- cells. These and other experiments show that PRPP is freely exchangeable between communication-competent cells and that the intracellular activity of HGPRTase in one cell can be regulated by changes in the levels of its substrate in another cell through metabolic cooperation. The results also indicate that HGPRTase normally functions at a small fraction of its total activity, and that this can be greatly increased by raising the intracellular PRPP levels. Furthermore, it is found that when communication-competent cells establish intercellular communication, they share a common pool of PRPP and of purine nucleotides. This approach can be used as the basis of a biochemical method for the quantitation of metabolic cooperation between cells.     

Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii

Inorganic pyrophosphate dependent D-fructose-6-phosphate 1-phosphotransferase from Propionibacterium freudenreichii was purified to apparent homogeneity by the criterion of silver staining on sodium dodecyl sulfate (SDS) gels. In the direction of phosphorylation of fructose 6-phosphate (F6P), an intersecting initial velocity pattern is obtained when MgPPi is varied at several levels of F6P. In the reverse reaction direction, the reactants are Mg2+, Pi, and fructose 1,6-bisphosphate (FDP). Variation of Pi at several levels of Mg2+ and a single level of FDP gives an intersecting pattern. When this pattern is repeated at several additional FDP levels, data are consistent with a fully random terreactant mechanism at pH 8.0 and 25 degrees C. The Keq calculated from the Haldane relationship [(5 +/- 1.5) X 10(-3) M] agrees with that determined directly from 31P NMR of the equilibrium mixture [(7 +/- 2) X 10(-3) M]. Product inhibition by Pi is competitive vs. either MgPPi or F6P with the other reactant saturating but changes to noncompetitive inhibition when the fixed reactant is decreased to Km levels. Product inhibition by MgPPi is competitive vs. either Pi or FDP with the other reactant saturating but changes to noncompetitive when the fixed reactant is decreased to Km levels. Tagatose 6-phosphate is competitive vs. F6P and noncompetitive vs. MgPPi. Methylenediphosphonate is competitive vs. MgPPi and noncompetitive vs. F6P. Sulfate is competitive vs. Pi and noncompetitive vs. FDP, while 2,5-anhydro-D-mannitol 1,6-bisphosphate is competitive vs. FDP and noncompetitive vs. Pi.     

Human placental adenosine kinase. Kinetic mechanism and inhibition

The kinetic properties of human placental adenosine kinase, purified 3600-fold, were studied. The reaction velocity had an absolute requirement for magnesium and varied with the pH. Maximal activity was observed at pH 6.5 with a Mg2+:ATP ranging from 1:1 to 2:1. High concentrations of Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both adenosine and MgATP2-. The Michaelis constant was 0.4 micro M for adenosine and 75 micro M for MgATP2-. Inhibition by adenosine was observed at concentrations greater than 2.5 micro M. AMP was a competitive inhibitor with respect to adenosine and a noncompetitive inhibitor with respect to ATP. ADP was a noncompetitive inhibitor with respect to adenosine and ATP. Hyperbolic inhibition was observed during noncompetitive inhibition of adenosine kinase by AMP and ADP. Other purine and pyrimidine nucleoside mono-, di-, and triphosphates were poor inhibitors in general. S-Adenosylhomocysteine and 2'-deoxyadenosine inhibited adenosine kinase. The data suggest that (a) MgATP2- is the true substrate of adenosine kinase, and both pH and [Mg2+] may regulate its activity; (b) the kinetic mechanisms of adenosine kinase is Ordered Bi Bi; and (c) adenosine kinase may be regulated by the concentrations of its products, AMP and ADP, but is relatively insensitive to other purine and pyrimidine nucleotides.     

Adenosinetriphosphate sulfurylase from Penicillium chrysogenum: steady-state kinetics of the forward and reverse reactions, alternative substrate kinetics, and equilibrium binding studies

The kinetics of the forward ATP sulfurylase-catalyzed reaction were examined using a new assay based on 32PPi released from [gamma-32P]MgATP in the presence of inorganic sulfate. Replots yielded Vmaxf = 6.6 units mg protein-1, KmA = 0.13 mM, Kia = 0.33 mM, and KmB = 0.55 mM, where A = MgATP and B = SO2-4. Thiosulfate, a dead-end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio kcat/KmA was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO2-4, SeO2-4, MoO2-4, WO2-4, or CrO2-4. For SO2-4 and SeO2-4, the ratio was 5-6.5 X 10(4) M-1 S-1; for the others, the ratio was 5.8-7.3 X 10(5) M-1 S-1. The results support a random addition of MgATP and inorganic substrate. The kinetics of the reverse reaction were examined using a new assay based on 35SO2-4 release from [35S]APS (adenosine 5'-phosphosulfate) in the presence of MgPPi. Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded Vmaxr = 50 units mg protein-1, KmQ = 0.3 microM, Kiq = 0.04 microM, and KmP = 4 microM, where Q = APS and P = PPi (total of all species). MgATP and SO2-4 were both competitive with APS and noncompetitive with respect to MgPPi. Taken together with earlier results suggesting that APS is competitive with both MgATP and SO2-4 and that MgPPi is noncompetitive with respect to both substrates, the qualitative results point to a random A-B, ordered P-Q kinetic mechanism. The Scatchard plot for [35S]APS binding was curved, indicating either negative cooperativity or more than a single class of sites. [gamma-32P]MgATP displayed half-site saturation in the presence of saturating FSO-3.     

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.     

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.     

Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization

The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin fibroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.     

Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.     

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

A point mutation at the subunit interface of hypoxanthine-guanine-xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from Plasmodium falciparum catalyzes the phosphoribosylation of hypoxanthine, guanine and xanthine. The functionally active form of HGXPRT is a tetramer but interface residues do not contribute to catalysis. Here we report the characterization of an interface mutant Y96C, which has a decreased k(cat), an increase in the K(m) for phosphoribosyl pyrophosphate (PRPP) and no change in K(m) for the purine bases when compared to the wild type enzyme. The mutant enzyme does not tetramerize in the presence of PRPP, unlike the wild type in which the tetramer is stabilized by PRPP. This is the first report of a HGXPRT mutation, at a unique interface where non-adjacent subunits interact, that impairs catalysis.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.