The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5′-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH‡) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.

The hydrophobicities of the 20 common amino acids are reflected in their tendencies to appear in interior positions in globular proteins and in deeply buried positions of membrane proteins. To determine whether these relationships might also have been valid in the warm surroundings where life may have originated, we examined the effect of temperature on the hydrophobicities of the amino acids as measured by the equilibrium constants for transfer of their side-chains from neutral solution to cyclohexane (Kw>c). The hydrophobicities of most amino acids were found to increase with increasing temperature. Because that effect is more pronounced for the more polar amino acids, the numerical range of Kw>c values decreases with increasing temperature. There are also modest changes in the ordering of the more polar amino acids. However, those changes are such that they would have tended to minimize the otherwise disruptive effects of a changing thermal environment on the evolution of protein structure. Earlier, the genetic code was found to be organized in such a way that—with a single exception (threonine)—the side-chain dichotomy polar/nonpolar matches the nucleic acid base dichotomy purine/pyrimidine at the second position of each coding triplet at 25 °C. That dichotomy is preserved at 100 °C. The accessible surface areas of amino acid side-chains in folded proteins are moderately correlated with hydrophobicity, but when free energies of vapor-to-cyclohexane transfer (corresponding to size) are taken into consideration, a closer relationship becomes apparent.

The hydrophobicities of the 20 common amino acids are reflected in their tendencies to appear in interior positions in globular proteins and in deeply buried positions of membrane proteins. To determine whether these relationships might also have been valid in the warm surroundings where life may have originated, we examined the effect of temperature on the hydrophobicities of the amino acids as measured by the equilibrium constants for transfer of their side-chains from neutral solution to cyclohexane (Kw>c). The hydrophobicities of most amino acids were found to increase with increasing temperature. Because that effect is more pronounced for the more polar amino acids, the numerical range of Kw>c values decreases with increasing temperature. There are also modest changes in the ordering of the more polar amino acids. However, those changes are such that they would have tended to minimize the otherwise disruptive effects of a changing thermal environment on the evolution of protein structure. Earlier, the genetic code was found to be organized in such a way that—with a single exception (threonine)—the side-chain dichotomy polar/nonpolar matches the nucleic acid base dichotomy purine/pyrimidine at the second position of each coding triplet at 25 °C. That dichotomy is preserved at 100 °C. The accessible surface areas of amino acid side-chains in folded proteins are moderately correlated with hydrophobicity, but when free energies of vapor-to-cyclohexane transfer (corresponding to size) are taken into consideration, a closer relationship becomes apparent.

Ever since the publication of Darwin’s Origin of Species, questions have been raised about whether enough time has elapsed for living organisms to have reached their present level of complexity by mutation and natural selection. More recently, it has become apparent that life originated very early in Earth’s history, and there has been controversy as to whether life originated in a hot or cold environment. This review describes evidence that rising temperature accelerates slow reactions disproportionately, and to a much greater extent than has been generally recognized. Thus, the time that would have been required for primordial chemistry to become established would have been abbreviated profoundly at high temperatures. Moreover, if the catalytic effect of a primitive enzyme (like that of modern enzymes) were to reduce a reaction’s heat of activation, then the rate enhancement that it produced would have increased as the surroundings cooled, quite aside from changes arising from mutation (which is itself highly sensitive to temperature). Some nonenzymatic catalysts of slow reactions, including PLP as a catalyst of amino acid decarboxylation, and the CeIV ion as a catalyst of phosphate ester hydrolysis, have been shown to meet that criterion. The work reviewed here suggests that elevated temperatures collapsed the time required for early evolution on Earth, furnishing an appropriate setting for exploring the vast range of chemical possibilities and for the rapid evolution of enzymes from primitive catalysts.

In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I–, Br–, Cl–, F–, HO–, and H2O in water at elevated temperatures. Comparison of the resulting second-order rate constants, extrapolated to 25 °C, with the values of kcat/Km reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 1015- and 1 × 1017-fold, respectively. These rate enhancements, achieved without the assistance of cofactors, metal ions, or general acid–base catalysis, are the largest that have been reported for an enzyme that acts on two substrates.

The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate, and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 1011. Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with catalytic rate accelerations of ∼104, whereas the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ∼1015. Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO2X–, are shown to be correlated by a two-parameter equation based on pKaArOH and pKaArOSO2XH.

To compare the powers of the corresponding enzymes as catalysts, the rates of uncatalyzed decarboxylation of several aliphatic acids (oxalate, malonate, acetoacetate, and oxaloacetate) were determined at elevated temperatures and extrapolated to 25 °C. In the extreme case of oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1 × 10−12 s−1, implying a 2.5 × 1013-fold rate enhancement by oxalate decarboxylase. Whereas the enzymatic decarboxylation of oxalate requires O2 and MnII, the uncatalyzed reaction is unaffected by the presence of these cofactors and appears to proceed by heterolytic elimination of CO2.

Experiments at elevated temperatures permit the determination of rate constant and thermodynamic activation parameters for the neutral hydrolysis of the neurotransmitter acetylcholine in water. At 25 °C, the extrapolated rate constant for the uncatalyzed (or neutral) hydrolysis of acetylcholine is 3.9 × 10–7 s–1 at 25 °C (ΔH‡ = 20.0 kcal/mol; TΔS‡ = −6.1 kcal/mol). Acetylcholine is more susceptible to neutral and base-catalyzed hydrolysis than ethyl acetate but less susceptible to acid-catalyzed hydrolysis. For acetylcholinesterase from the electric eel, the catalytic proficiency [(kcat/Km)/kneutral] is 2 × 1016 M–1, comparable in magnitude with the catalytic proficiencies of aminohydrolases that act on peptides and nucleosides.

The hydrolysis of phosphate diesters is one of the most difficult reactions known. Here we show that in acetone or cyclohexane, at 25 °C, phosphodiesters undergo hydrolysis 5 × 105 and 2 × 109-fold more rapidly than in water, respectively, and that this rate enhancement is achieved by lowering the enthalpy of activation.

Previous estimates of the rate of spontaneous cleavage of the glycosidic bond of adenosine were determined by extrapolating the rates of the acid- and base-catalyzed reactions to neutral pH. Here we show that cleavage also proceeds through a pH-independent mechanism. Rate constants were determined as a function of temperature at pH 7 and a linear Arrhenius plot was constructed. Uncatalyzed cleavage occurs with a rate constant of 3.7 × 10−12 s−1 at 25 °C, and the rate enhancement generated by the corresponding glycoside hydrolase is ∼5 × 1012-fold.We show that hydrolysis of the glycosidic CN bond of adenosine, previously known to be subject to hydrolysis in acid and base, also proceeds through a pH-independent mechanism with a rate constant of 4 × 10−12 s−1 at 25 °C.

All reactions are accelerated by an increase in temperature, but the magnitude of that effect on very slow reactions does not seem to have been fully appreciated. The hydrolysis of polysaccharides, for example, is accelerated 190,000-fold when the temperature is raised from 25 to 100 °C, while the rate of hydrolysis of phosphate monoester dianions increases 10,300,000-fold. Moreover, the slowest reactions tend to be the most heat-sensitive. These tendencies collapse, by as many as five orders of magnitude, the time that would have been required for early chemical evolution in a warm environment. We propose, further, that if the catalytic effect of a “proto-enzyme”—like that of modern enzymes—were mainly enthalpic, then the resulting rate enhancement would have increased automatically as the environment became cooler. Several powerful nonenzymatic catalysts of very slow biological reactions, notably pyridoxal phosphate and the ceric ion, are shown to meet that criterion. Taken together, these findings greatly reduce the time that would have been required for early chemical evolution, countering the view that not enough time has passed for life to have evolved to its present level of complexity.

The hydrolysis of simple phosphate monoesters is among the most difficult reactions that are subject to catalysis by enzymes, and it has been suggested that extraction of the substrates from solvent water may contribute to the catalytic effects of phosphohydrolases. Here, we show that the tetrabutylammonium salt of neopentyl phosphate enters wet cyclohexane at concentrations sufficient to allow determination of its rate of hydrolysis. The second-order rate constant for hydrolysis of the phosphomonoester dianion is enhanced ∼2 × 1012-fold by transfer from water to cyclohexane. That rate enhancement arises from an increase in the entropy of activation.

OMP decarboxylase (ODCase) generates a very large rate enhancement without the assistance of metals or other cofactors. The uncatalyzed decarboxylation of 1-methylorotate in water is shown to involve the monoanion, although uncharged 1-methylorotic acid is decarboxylated at a similar rate. To measure the extent to which the rate of the nonenzymatic decarboxylation of orotate derivatives might be enhanced by their removal from solvent water, the 1-phosphoribosyl moiety of OMP was replaced with 1-substituents that would allow it to enter less polar solvents. When the tetrabutylammonium salt of 1-cyclohexylorotate was transferred from water to a series of dipolar aprotic solvents, its rate of decarboxylation increased markedly, varying with the relative ability of each solvent to release the substrate in the ground state from stabilization by solvent water acting as a proton donor. These findings are consistent with the view that separation of the substrate from solvent water may contribute, at least to a limited extent, to the rate enhancement produced by ODCase. This enzyme’s active site, like that of another cofactorless enzyme recently shown to produce a rate enhancement similar in magnitude (uroporphyrinogen decarboxylase), is equipped with an ammonium group positioned in such a way as to balance the electrostatic charge of the carboxylate group of the substrate and later supply a proton to the incipient carbanion in a relatively waterless environment.

Alkyl sulfate monoesters are involved in cell signaling and structure. Alkyl sulfates are also present in many commercial detergents. Here, we show that monomethyl sulfate acts as an efficient alkylating agent in water, reacting spontaneously with oxygen nucleophiles >100-fold more rapidly than do alkylsulfonium ions, the usual methyl donors in living organisms. These reactions of methyl sulfate, which are much more rapid than its hydrolysis, are insensitive to the nature of the attacking nucleophile, with a Brønsted β_nuc value of −0.01. Experiments at elevated temperatures indicate a rate constant of 2 × 10⁻¹¹ s⁻¹ for the uncatalyzed hydrolysis of methyl sulfate at 25°C (t _1/2 = 1,100 y), corresponding to a rate enhancement of ≈10¹¹-fold by a human alkylsulfatase. Equilibria of formation of methyl sulfate from methanol and sodium hydrogen sulfate indicate a group transfer potential (ΔG′ _pH7) of −8.9 kcal/mol for sulfate ester hydrolysis. The magnitude of that value, involving release of the strong acid HSO₄ ⁻, helps to explain the need for harnessing the free energy of hydrolysis of two ATP molecules in activating sulfate for the biosynthesis of sulfate monoesters. The “energy-rich” nature of monoalkyl sulfate esters, coupled with their marked resistance to hydrolysis, renders them capable of acting as sulfating or alkylating agents under relatively mild conditions. These findings raise the possibility that, under appropriate circumstances, alkyl groups may undergo transfer from alkyl sulfate monoesters to biological target molecules.

Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C–O cleavage, at a rate suggesting an upper limit of 10−15 s−1 for P–O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P–O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P–O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C–O cleavage is sterically precluded so that hydrolysis occurs only by P–O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t 1/2 of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k cat = 95 s−1) enhances the rate of phosphodiester hydrolysis by a factor of ≈1017. In alkaline solution, thymidylyl-3′-5′-thymidine (TpT) has been reported to decompose 105-fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.

The soil of potato fields in The Netherlands harbors bacteria with the ability to metabolize 3-chloroacrylic acid, generated by the degradation of a pesticide (1,3-dichloropropene) that entered the environment in 1946. From examination of rate constants at elevated temperatures, we infer that the half-time at 25°C for spontaneous hydrolytic dechlorination of trans-3-chloroacrylic acid is 10,000 years, several orders of magnitude longer than half-times for spontaneous decomposition of other environmental pollutants such as 1,2-dichloroethane (72 years), paraoxon (13 months), atrazine (5 months), and aziridine (52 h). With thermodynamic parameters for activation similar to those for the spontaneous hydration of fumarate at pH 6.8, this slow reaction proceeds at a constant rate through the pH range between 2 and 12. However, at the active site of the enzyme 3-chloroacrylate dehalogenase (CaaD), isolated from a pseudomonad growing in these soils, hydrolytic dechlorination proceeds with a half-time of 0.18 s. Neither k cat nor k cat/K m is reduced by increasing solvent viscosity with trehalose, implying that the rate of enzymatic dechlorination is controlled by chemical events in catalysis rather than by diffusion-limited substrate binding or product release. CaaD achieves an ≈1012-fold rate enhancement, matching or surpassing the rate enhancements produced by many enzymes that act on more conventional biological substrates. One of those enzymes is 4-oxalocrotonate tautomerase, with which CaaD seems to share a common evolutionary origin.

The soil of potato fields in The Netherlands harbors bacteria with the ability to metabolize 3-chloroacrylic acid, generated by the degradation of a pesticide (1,3-dichloropropene) that entered the environment in 1946. From examination of rate constants at elevated temperatures, we infer that the half-time at 25°C for spontaneous hydrolytic dechlorination of trans-3-chloroacrylic acid is 10,000 years, several orders of magnitude longer than half-times for spontaneous decomposition of other environmental pollutants such as 1,2-dichloroethane (72 years), paraoxon (13 months), atrazine (5 months), and aziridine (52 h). With thermodynamic parameters for activation similar to those for the spontaneous hydration of fumarate at pH 6.8, this slow reaction proceeds at a constant rate through the pH range between 2 and 12. However, at the active site of the enzyme 3-chloroacrylate dehalogenase (CaaD), isolated from a pseudomonad growing in these soils, hydrolytic dechlorination proceeds with a half-time of 0.18 s. Neither k cat nor k cat/K m is reduced by increasing solvent viscosity with trehalose, implying that the rate of enzymatic dechlorination is controlled by chemical events in catalysis rather than by diffusion-limited substrate binding or product release. CaaD achieves an ≈1012-fold rate enhancement, matching or surpassing the rate enhancements produced by many enzymes that act on more conventional biological substrates. One of those enzymes is 4-oxalocrotonate tautomerase, with which CaaD seems to share a common evolutionary origin.

The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group). To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode. Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase. In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule.

To determine the effectiveness of the ribosome as a catalyst, we compared the rate of uncatalyzed peptide bond formation, by the reaction of the ethylene glycol ester of N-formylglycine with Tris(hydroxymethyl)aminomethane, with the rate of peptidyl transfer by the ribosome. Activation parameters were also determined for both reactions, from the temperature dependence of their second-order rate constants. In contrast with most protein enzymes, the enthalpy of activation is slightly less favorable on the ribosome than in solution. The 2 × 107-fold rate enhancement produced by the ribosome is achieved entirely by lowering the entropy of activation. These results are consistent with the view that the ribosome enhances the rate of peptide bond formation mainly by positioning the substrates and/or water exclusion within the active site, rather than by conventional chemical catalysis.

The hydrolysis of phosphate diesters is one of the most difficult reactions known. Here we show that in acetone or cyclohexane, at 25 °C, phosphodiesters undergo hydrolysis 5 × 105 and 2 × 109-fold more rapidly than in water, respectively, and that this rate enhancement is achieved by lowering the enthalpy of activation.