At the active site of urease, urea undergoes nucleophilic attack by water, whereas urea decomposes in solution by elimination of ammonia so that its rate of spontaneous hydrolysis is unknown. Quantum mechanical simulations have been interpreted as indicating that urea hydrolysis is extremely slow, compared with other biological reactions proceeding spontaneously, and that urease surpasses all other enzymes in its power to enhance the rate of a reaction. We tested that possibility experimentally by examining the hydrolysis of 1,1,3,3-tetramethylurea, from which elimination cannot occur. In neutral solution at 25 degrees C, the rate constant for the uncatalyzed hydrolysis of tetramethylurea is 4.2 x 10-12 s-1, which does not differ greatly from the rate constants observed for the uncatalyzed hydrolysis of acetamide (5.1 x 10-11 s-1) or N,N-dimethylacetamide (1.8 x 10-11 s-1) under the same conditions. We estimate that the proficiency of urease as a catalyst, (kcat/Km)/knon, is 8 x 1017 M-1, slightly higher than the values for other metalloenzymes (carboxypeptidase b and cytidine deaminase) that catalyze the hydrolysis of similar bonds.

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-tocyclohexane transfer (corresponding to size) are taken into consideration, a closer relationship becomes apparent.T he equilibrium conformations of proteins in neutral solution are strongly influenced by interactions between their constituent amino acids and solvent water. Early work on the crystal structure of hemoglobin and related proteins showed that the side-chains of the more polar amino acid residues tend to be exposed to solvent, whereas less polar side-chains tend to be buried within the interior of globular proteins (1). Later, those tendencies were put to a quantitative test by measuring equilibria of transfer of amino acid side-chains from neutral aqueous solution into less polar environments, such as the vapor phase (2, 3) or a nonpolar solvent such as cyclohexane (4), which dissolves only ∼2 × 10 −3 M water at saturation (5) and appears to be devoid of specific interactions with solutes. The water-to-cyclohexane distribution coefficients (K w>c ) of the 20 common sidechains [here termed "hydrophobicities" (6, 7) and expressed in concentration units of mol/L in each phase; SI Appendix] were found to span a range of 15 orders of magnitude at pH 7 and 25°C. Values of K w>c have been shown to be related to their outside-to-inside distributions in globular proteins (4,8) and to their tendencies to appear within the buried sequences of transmembrane proteins (9-11).Those solvent distribution experiments were conducted at what we would consider ordinary temperatures. However, there is widespread (12, 13)-if not universal (14)-agreement that life originated when the ea...

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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.activation energy | thermophilic organisms | pyridoxal phosphate | phosphate ester hydrolysis | amino acid decarboxylation W hereas enzyme reactions ordinarily occur in a matter of milliseconds, the same reactions proceed with half-lives of hundreds, thousands, or millions of years in the absence of a catalyst ( Fig. 1) (1). Yet life is believed to have taken hold within the first 25% of Earth's history (2). How could cellular chemistry, and the enzymes that make life possible, have arisen so quickly? Here, we show that because of an extraordinarily sensitive relationship between temperature and the rates of very slow reactions, the time required for early evolution on a warm earth was very much shorter than it might appear. That sensitivity also suggests some likely properties of an evolvable catalyst, and a testable mechanism by which its ability to enhance rates might have been expected to increase as the environment cooled.Rapid substrate turnover is necessary to support the metabolism of an organism at the enzyme concentrations found in cells, but the same reactions, in the absence of enzymes, proceed vastly more slowly (Fig. 1). For example, the decarboxylation of orotidine 5′-phosphate (OMP), the final step in the biosynthesis of pyrimidines-and thus nucleic acids-proceeds with a half-life of 0.017 s at the active site of OMP decarboxylase. In neutral solution in the absence of the enzyme, the same reaction proceeds with a half-life of 78 million years (1). It is natural to ask how enzymes arose to meet so formidable a challenge. The Time Required for Primordial Chemistry to Become EstablishedThe rates of simple reactions, even if they are immeasurably slow at ordinary temperatures, can often be estimated by first determining their rates at elevated temperatures. Plots of the logarithm of the observed rate constants a...

The half-lives for spontaneous hydrolysis of trehalose and sucrose at 25 °C are 6.6 × 106 years and 440 years. The half-lives for decomposition of the hydrolysis products glucose and fructose are 96 years and 70 days, respectively. Whereas sucrose and trehalose differ by a factor of 15000 in their rates of uncatalyzed hydrolysis, the reactions catalyzed by invertase (EC 3.2.1.26) and trehalase (EC 3.2.1.28) proceed at similar rates. Accordingly, the attainments of invertase as a catalyst are modest, but the rate enhancement and catalytic proficiency produced by trehalase approach the high levels achieved by polysaccharide hydrolases.