Benchmark reaction rates, the stability of biological molecules in water, and the evolution of catalytic power in enzymes.

The rates of enzyme reactions fall within a relatively narrow range. To estimate the rate enhancements produced by enzymes, and their expected affinities for transition state analog inhibitors, it is necessary to measure the rates of the corresponding reactions in water in the absence of a catalyst. This review describes the spontaneous cleavages of C-C, C-H, C-N, C-O, P-O, and S-O bonds in biological molecules, as well as the uncatalyzed reactions that correspond to phosphoryl transfer reactions catalyzed by kinases and to peptidyl transfer in the ribosome. The rates of these reactions, some with half-lives in excess of one million years, span an overall range of 10¹9-fold. Moreover, the slowest reactions tend to be most sensitive to temperature, with rates that increase as much as 107-fold when the temperature is raised from 25° to 100°C. That tendency collapses, by many orders of magnitude, the time that would have been required for chemical evolution on a warm earth. If the catalytic effect of primitive enzymes, like that of modern enzymes and many nonenzymatic catalysts, were mainly to reduce a reaction's enthalpy of activation, then the resulting rate enhancement would have increased automatically as the surroundings cooled. By reducing the time required for early chemical evolution in a warm environment, these findings counter the view that not enough time has passed for terrestrial life to have evolved to its present level of complexity.

Aldol Cleavage in Water and the Power of Citrate Lyase as a Catalyst.

Citrate lyase allows Klebsiella aerogenes to grow anaerobically on citrate as the sole carbon source. Arrhenius analysis of experiments at high temperatures indicates that citrate is cleaved nonenzymatically to acetate and oxaloacetate with a t1/2 of 6.9 million years in neutral solution at 25 °C, while malate cleavage occurs even more slowly (t1/2 = 280 million years). However, t1/2 is only 10 days for the nonenzymatic cleavage of 4-hydroxy-2-ketoglutarate, indicating that the introduction of an α-keto group enhances the rate of aldol cleavage of malate by a factor of 1010. The aldol cleavages of citrate and malate, like the decarboxylation of malonate (t1/2 = 180 years), are associated with a near-zero entropy of activation, and their extreme differences in rate reflect differences between their heats of activation. Citrate lyase enhances the rate of substrate cleavage 6 × 1015-fold, comparable in magnitude with the rate enhancement produced by OMP decarboxylase, although these enzymes are strikingly different in their mechanisms of action.

The Burden Borne by Protein Methyltransferases: Rates and Equilibria of Non-enzymatic Methylation of Amino Acid Side Chains by SAM in Water.

SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of the equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with an increase in pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary, and quaternary amines are found to be insensitive to a change in pH and resemble each other in magnitude, with an average ΔG value of approximately -0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono-, di-, and trimethylation of lysine by SAM is accompanied by a change in free energy of -7.5 kcal/mol in a neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+) with second-order rates constant of 1.8 × 10-7 M-1 s-1 at 25 °C (ΔH⧧ = 22 kcal/mol, and TΔS⧧ = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k25 values of 1.1 × 10-7 M-1 s-1 for sarcosine and 4.3 × 10-8 M-1 s-1 for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS+, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k25 values of 3.3 × 10-9 and 1.2 × 10-9 M-1 s-1, respectively. Lysine methylation by SAM, although slow under physiological conditions (t1/2 = 7 weeks at 25 °C), is accelerated 1.1 × 1012 -fold at the active site of a SET domain methyltransferase.

Ether Hydrolysis, Ether Thiolysis, and the Catalytic Power of Etherases in the Disassembly of Lignin.

The recycling of much of the carbon in Nature depends on the breakdown of polymers in woody matter, notably cellulose (a polyacetal) and lignin (a polyether). Here, we show that equilibrium favors ether hydrolysis in water, although the rates of spontaneous hydrolysis of ethers are too slow to measure in neutral solution except at temperatures approaching the critical point of water. Circumventing that kinetic obstacle, glutathione-dependent etherases from white-rot fungi are known to employ the thiolate group of glutathione to attack guaiacyl ethers. Experiments at elevated temperatures indicate that thioglycolate attacks diethyl ether in water, in the absence of enzymes, with a rate constant of 6 × 10-11 M-1 s-1 at 25 °C and that ether thiolysis is strongly favored thermodynamically, with a Keq value of 2.5 × 106 (ΔG = -8.7 kcal/mol). Compared with the rate of non-enzymatic thiolysis, the lignin-degrading etherases LigE and LigF produce 1015-fold rate enhancements, among the largest that have been observed for an enzyme acting on two substrates.

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

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.

Sulfonium Ion Condensation: The Burden Borne by SAM Synthetase.

S-Adenosylmethionine (SAM+) serves as the principal methylating agent in biological systems, but the thermodynamic basis of its reactivity does not seem to have been clearly established. Here, we show that methionine, methanol, and H+ combine to form S-methylmethionine (SMM+) with a temperature-independent equilibrium constant of 9.9 M-2. The corresponding group transfer potential of SMM+ (its free energy of hydrolysis at pH 7) is -8.2 kcal/mol. The "energy-rich" nature of sulfonium ions is related to the extreme acidity (p Ka -5.4) of the S-protonated thioether produced by sulfonium hydrolysis, and the large negative free energy of deprotonation of that species in neutral solution (-16.7 kcal/mol). At pH 7, SAM synthetase requires the free energy released by cleavage of two bonds of ATP to reverse that process.

tRNA acceptor stem and anticodon bases form independent codes related to protein folding.

Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3' acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each side-chain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for ß-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or ß-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water.

Temperature dependence of amino acid hydrophobicities.

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 (K(w > 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 K(w > 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.

Three Pyrimidine Decarboxylations in the Absence of a Catalyst.

The epigenetic modification of DNA by 5-methylation of cytosine residues can be reversed by the action of the TET family of dioxygenases that oxidize the methyl group to produce 5-carboxycytosine (5caC), which can be converted to cytosine in a final decarboxylation step. Likewise, 5-carboxyuracil (5caU) is decarboxylated to uracil in the last step in pyrimidine salvage. In view of the extreme difficulty of decarboxylating derivatives of orotic acid (6caU), it seemed desirable to establish the rates of decarboxylation of 5caC and 5caU in the absence of a catalyst. Arrhenius analysis of experiments performed at elevated temperatures indicates that 5caU decomposes with a rate constant of 1.1 × 10-9 s-1 (ΔH⧧ = 25 kcal/mol) in a neutral solution at 25 °C. The decomposition of 5caC is somewhat slower (k25 = 5.0 × 10-11 s-1; ΔH⧧ = 27 kcal/mol) and leads to the initial accumulation of cytosine as an intermediate, followed by the relatively rapid deamination of cytosine (k25 = 1.9 × 10-10 s-1; ΔH⧧ = 23.4 kcal/mol). Both 5caC and 5caU are decarboxylated many orders of magnitude more rapidly than 6caU is (k25 = 1.3 × 10-17 s-1). Ab initio simulations indicate that in all three cases, the favored route of spontaneous decarboxylation in water involves direct elimination of CO2 with the assistance of an explicit water molecule.

The triple threat of HIV-1 protease inhibitors.

Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy.

tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry.

The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology.

Massive thermal acceleration of the emergence of primordial chemistry, the incidence of spontaneous mutation, and the evolution of enzymes.

Kelvin considered it unlikely that sufficient time had elapsed on the earth for life to have reached its present level of complexity. In the warm surroundings in which life first appeared, however, elevated temperatures would have reduced the kinetic barriers to reaction. Recent experiments disclose the profound extent to which very slow reactions are accelerated by elevated temperatures, collapsing the time that would have been required for early events in primordial chemistry before the advent of enzymes. If a primitive enzyme, like model catalysts and most modern enzymes, accelerated a reaction by lowering its enthalpy of activation, then the rate enhancement that it produced would have increased automatically as the environment cooled, quite apart from any improvements in catalytic activity that arose from mutation and natural selection. The chemical events responsible for spontaneous mutation are also highly sensitive to temperature, furnishing an independent mechanism for accelerating evolution.

Primordial chemistry and enzyme evolution in a hot environment.

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 Ce(IV) 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.

The nonenzymatic decomposition of guanidines and amidines.

To establish the rates and mechanisms of decomposition of guanidine and amidine derivatives in aqueous solution and the rate enhancements produced by the corresponding enzymes, we examined their rates of reaction at elevated temperatures and used the Arrhenius equation to extrapolate the results to room temperature. The similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is consistent with attack by hydroxide ion on the methylguanidinium ion (k2 = 5 × 10(-6) M(-1) s(-1) at 25 °C) or with the kinetically equivalent attack by water on uncharged methylguanidine. At 25 °C and pH 7, N-methylguanidine is several orders of magnitude more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate substrate hydrolysis 4 × 10(14)-fold and 6 × 10(12)-fold, respectively, by mechanisms that appear to involve metal-mediated water attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10(12)-fold by a mechanism that (in contrast to the mechanisms employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.

Kinetic mechanism of human histidine triad nucleotide binding protein 1.

Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 10(6)-10(8)-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.

Catalysis by desolvation: the catalytic prowess of SAM-dependent halide-alkylating enzymes.

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 k(cat)/K(m) reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 10(15)- and 1 × 10(17)-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.

Impact of temperature on the time required for the establishment of primordial biochemistry, and for the evolution of enzymes.

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.

Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule.

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K(d) ~ 10(-12) M), capturing all but ~5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K(tx) ~ 10(-16) M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme-inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364-11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E(2)Zn(2)W(2) (dimeric Zn-CDA with two water molecules), E(2)Zn(2)W(2)Fz, and E(2)Zn(2)W(2)Fz(2), where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E(2)Zn(2) is the only dimeric species detected, with no additional water molecules. Experiments conducted in H(2)(18)O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E(2)Zn(2)) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k(off) > 0.14 s(-1)) much more rapidly than is 5-fluorozebularine (k(off) = 0.014 s(-1)), despite the greater thermodynamic stability of the zebularine complex.

Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase.

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, (31)P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP(i)) in the presence and absence of Mg(2+) at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP(i)(2-) proceeds with a rate constant of 2.8 × 10(-10) s(-1), whereas E. coli pyrophosphatase was found to have a turnover number of 570 s(-1) under the same conditions. The resulting rate enhancement (2 × 10(12)-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH(‡) = -16.6 kcal/mol). The presence of Mg(2+) ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼ 10(6)-fold. Transfer to dimethyl sulfoxide accelerated PP(i) hydrolysis by reducing the enthalpy of activation. Mg(2+) increased the rate of PP(i) hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.

Catalytic proficiency: the extreme case of S-O cleaving sulfatases.

As benchmarks for judging the catalytic power of sulfate monoesterases, we sought to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by S-O bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to undergo hydrolysis by a C-O bond cleaving mechanism with rearrangement of its carbon skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of aryl sulfate monoesters, which proceed by S-O cleavage. Extrapolation of a Bronsted plot [log(k(25)(N)) = (-1.81 ± 0.09) pK(a)(LG) + (3.6 ± 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate monoesters to a pK(a)(LG) value of 16.1, typical of an aliphatic alcohol, yields k(25)(N) = 3 × 10(-26) s(-1). Comparison of that value with established k(cat) values of bacterial sulfatases indicates that these enzymes produce rate enhancements (k(cat)/k(uncat)) of up to 2 × 10(26)-fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ~10(21)-fold rate enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of similar reactivity is dispelled.