Interaction of the high-affinity inhibitor tetrahydro-dUMP with the allosteric enzyme deoxycytidylate aminohydrolase.

Tetrahydro-dUMP, an analog of the putative transition state in aminohydrolysis of deoxycytidine monophosphate (dCMP) inhibits the allosteric enzyme deoxycytidylate aminohydrolase with high affinity. The inhibition is reversible, and its kinetics is consistent with the analog binding at the substrate site only to one and the same conformation that binds the substrate dCMP. Such kinetics is what would be expected for a transition state analog interacting in an allosteric "K system."

Hill coefficient ratios give binding ratios of allosteric enzyme effectors; inhibition, activation, and squatting in deoxycytidylate aminohydrolase (EC 3.5.4.12)

The ratio of the steady-state kinetic Hill coefficients of two different effectors equals (under some rather weak general assumptions) the ratio in which the effectors displace each other from an enzyme. This principle can make implications of experimental allosteric enzyme kinetic data immediately apparent. We can use it to find that one molecule of the allosteric inhibitor of dCMP aminohydrolase, at moderately high effector concentrations, displaces one molecule of substrate, or one molecule of activator, whereas at very high concentrations, one molecule of inhibitor displaces two of substrate. Further use of the principle suggests that substrate, at high concentrations, binds binds to activator sites. However, ratios of substrate, activator, and inhibitor Hill coefficients are incompatible with a simple model of activation in which substrate and activator are bound to the same conformation.

Allosteric modifier and substrate binding of donkey deoxycytidylate aminohydrolase (EC 3.5.4.12)

The hexameric allosteric enzyme deoxycytidylate aminohydrolase from donkey spleen is shown by equilibrium dialysis to bind specifically the allosteric inhibitor, dTTP, the activator dCTP, and the substrate analog dAMP each at six sites (the dTTP and dCTP sites may or may not be identical). These conclusions contrast with earlier ones that there were four sites for each effector; reasons for the discrepancy are discussed. With the knowledge of site numbers and the kinetic information from the accompanying paper it is concluded that the kinetic cooperativity of the enzyme excludes a concerted conformational transition mechanism. Amino acid analysis gives a molecular weight of 18,842 Da per subunit, i.e., 113,052 for the hexamer. A new simplified purification of homogeneous enzyme from donkey spleen probably useful for dCMP aminohydrolase from other sources is described.

Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.

Measurement of DNA methylase activity by tritium release from DNA cytosine.

The advantages of assaying of DNA methylase by measuring the transfer to water of tritium from the 5 position of DNA cytosine, rather than the transfer to DNA of labeled methyl groups are discussed.

Nonequivalence of the two subunits of horse erythrocyte glutathione transferase in their reaction with sulfhydryl reagents.

Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activity. A peculiar feature of these protein thiol groups is that they react nonidentically with a number of thiol blocking reagents, i.e. iodacetamide, bromopyruvate, N-ethylmaleimide, and 1-chloro-2,4-dinitrobenzene. Also many disulfides react with one thiol group 5- to 10-fold more rapidly than with the other. The two mixed disulfides so formed also have different rates of reactivation by dithiothreitol. All the structural and kinetic data reported in this paper indicate a nonsymmetrical association of two identical subunits, or alternatively heterodimeric structure with subunits of very similar charge and size.

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA.

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.

Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides.

A DNA methyltransferase partly purified from human placenta has been tested on a variety of synthetic polydeoxynucleotides. The results showed that: the enzyme is most active as a 'maintenance' or 'hemi-' methylase but also has some de novo methylating activity; the presence or absence of A or T in the substrate strand has little influence on maintenance or de novo activity, while polymers containing C but not G in the same strand are poor de novo substrates and bind poorly to the enzyme; single-stranded polymers are about as good substrates as double-stranded ones, and the effects of nucleotide composition (particularly G and mC content) on enzyme activity with single strands are similar to those with double-stranded polymers; strands in which all the cytosines are methylated bind the enzyme well. A mechanism is suggested involving two different sites on the enzyme that recognize CG and mCG, and which rationalizes the activity of eukaryotic DNA methyltransferases towards single-stranded DNA.

Hypomethylation of repair patches in HeLa cells.

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.

Analysis of competition for substrate sites in an allosteric enzyme with co-operative kinetics. Effects of dAMP and dUMP on donkey spleen deoxycytidylate aminohydrolase.

Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co-operative kinetics can be tested simply and exactly without knowledge of the nature of the intersubunit co-operativity by using a linkage approach to the analysis of steady-state kinetics. Applying this approach to competition for substrate sites in the allosteric enzyme donkey spleen dCMP aminohydrolase, we show that the kinetics are consistent with the hypothesis that the substrate dCMP and the competitor dAMP, as well as the allosteric activator dCTP, bind exclusively to the same conformation of the enzyme subunits. The linkage test can be applied in the presence of other effectors without knowledge of how these interact with the enzyme. Our tests showed that dCMP and DAMP are still bound exclusively to this same conformation in the presence of the product dUMP or of the allosteric inhibitor dTTP. We give evidence that dUMP binds to the same conformation as dCMP, but that it is also bound to other conformation(s). The advantages of the linkage approach, and some general problems in steady-state kinetics of allosteric enzymes, are discussed.

ATP-dependent exonuclease V from Micrococcus luteus. The enzyme-DNA complex, the processive mechanism, and the role of ATP.

Some kinetic predictions of the proposed processive mechanism for the hydrolysis of DNA by the ATP-dependent enzyme exonuclease V have been checked. The method is to trap enzyme molecules not attached to radioactive DNA substrate with an excess of nonradioactive DNA, so that enzyme molecules attached to the radioactive substrate contribute to the liberation of radioactive products only until they dissociate from it. The experiments show that enzyme molecules remain attached to a T7 double-stranded DNA molecule, while hydrolysing it, for about 2 min under our conditions, in agreement with the predictions of the processive mechanism. However, the mechanism of degradation of single-stranded DNA is not processive. Formation of an enzyme-DNA complex is largely dependent on the presence of ATP. This formation does not appear to be synchronous. ATP analogs do not stimulate formation of, nor stabilize, the enzyme-DNA complex. EDTA causes dissociation of enzyme molecules from the DNA complex.

Patterns of apparent co-operativity in a simple random non-equilibrium enzyme--substrate--modifier mechanism. Comparison with equilibrium allosteric models.

It has often been claimed that random non-equilibrium mechanisms can result in apparent homotropic and heterotropic effects in steady-state kinetics of the kind more usually attributed to intersubunit allosteric interactions. However, it has never been shown whether any simple random mechanism could in fact give patterns of apparent interaction similar to those predicted by the well-known allosteric models. The patterns of apparent substrate co-operativity and affinity given by the steady-state of a standard simple random substrate-modifier mechanism in which catalytic velocity is proportional to substrate binding have been analysed mathematically and numerically. All patterns possible with this model are described. Some of them rather resemble those possible with standard allosteric models, in that there is a high-affinity and a low-affinity form at zero and infinite modifier concentrations (or vice versa) which show Michaelian behaviour, apparent co-operativity passing through a maximum or minimum at intermediate affinities. Unlike the allosteric models the family of curves is in principle not symmetrical. The random model can also give behaviour not possible with the standard allosteric models, such as higher substrate affinity at intermediate modifier concentrations than at either zero or infinite modifier, with concomitant negative apparent substrate co-operativity, or a single change of sign of apparent substrate co-operativity. The analysis uses recently discovered simplified forms of steady-state equations for random models.

Co-operativity and the methods of plotting binding and steady-state kinetic data.

The features that distinguish positive from negative co-operativity in double-reciprocal Eadie-Hofstee-Scatchard and Hanes plots, often incorrectly stated to be the sign of curvature or second derivatives, are explained. It is shown how to determine the 'Hill exponent' and interaciton free energies from curves in these plots, and in the simple plot of ligand binding or velocity against free ligand or substrate concentration. New types of plots, where the kind of co-operative behaviour is more obvious than in the traditional ones, are proposed.

Simplifications of the derivations and forms of steady-state equations for non-equilibrium random substrate-modifier and allosteric enzyme mechanisms.

The steady-state equations for "random" enzymic mechanisms (ones with alternative routes for substrate and enzyme to form enzyme-substrate complexes) are non-Michaelian and very complicated when a quasi-equilibrium approximation cannot be used. General methods for simplifying their forms and derivations are given and applied to several single-substrate mechanisms of general or topical interest. The special simplifications resulting from partial ordering of reaction mechanism, from gross inequalities of rate constants, and from special relationships between catalytic and dissociation rate constants, are considered with reference to allosteric mechanisms. Some equations mentioned, but not given here, and more detailed working out of some of those given, have been deposited as Supplementary Publication SUP 50069 (18 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153, 5.

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe.

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.