Rates of Spontaneous Cleavage of Glucose, Fructose, Sucrose, and Trehalose in Water, and the Catalytic Proficiencies of Invertase and Trehalas

Most of the early experimental work on enzyme kinetics was based on the properties of yeast invertase (-fructo-furanosidase, EC 3.2.1.26), whose robust activity1 (together with the availability of a continuous polarimetric assay) enabled Michaelis and Menten to establish the existence of a quantitative relationship between the rate of an enzyme reaction and the concentration of its substrate.2 To appreciate the proficiency of an enzyme as a catalyst, it is also desirable to have information about the relative rates of the catalyzed and uncatalyzed reactions.3 Surprisingly, the literature discloses no report of the rate of spontaneous hydrolysis of sucrose (although there have been many studies of the acid-catalyzed reaction).4 That information would be of special interest in view of the remarkable resistance to hydrolysis (t1/2 ∼107 y),5 of the 1-4 “head-to-tail” glycosidic linkages that join the common glucose polymers cel-lulose, chitin, amylose, and glycogen. Thus, polysaccharide hy

Experimental Measures of Amino Acid Hydrophobicity and the Topology of Transmembrane and Globular Proteins

Roles of Solvent Water in the Positions of Biological Equilibria The noncovalent binding interactions of biological molecules involve the stripping away of solvent water from regions of contact between the binding partners. Accordingly, the net strength of their interactions with other molecules can be considered to include the cost of removing the interacting molecules (at least those parts that make contact with each other) from the sol- vent to which they were previously exposed. Free ener- gies of solvation of biological molecules also play a major role in determining the positions of their chemi- cal equilibria. For example, the products of hydrolysis of ATP are so much more strongly solvated than the re- actants that changing solvation fully accounts for the favorable equilibrium of hydrolysis of ATP to ADP and inorganic phosphate (Williams and Wolfenden, 1985). It has long been suspected that solvent water plays a major role in the equilibrium structure of globular pro- teins in solution, which typically present the aspect of "an oil drop with a polar coat" (Kauzmann, 1959). Com- pared with the average environment experienced by an amino acid residue in the interior of a globular protein, an amino acid side chain's environment in the interior of a biological membrane is probably relatively isomor- phous. The penetrating experiments of von Heijne and his associates (Hessa et al., 2005) have furnished quan- titative information about the relative tendencies of the various amino acids to be found in deeply immersed re- gions of membrane proteins, and an opportunity to compare these tendencies with solvation properties of the amino acids. Those latter properties are the subject of this commentary. Amino acids: The Choice of a Representative Solut

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 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

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

The universal genetic code is the earliest point to which we can trace biological inheritance. Earlier work hinted at a relationship between the codon bases and the physical properties of the 20 amino acids that dictate the 3D conformations of proteins in solution. Here, we show that acceptor stems and anticodons, which are at opposite ends of the tRNA molecule, code, respectively, for size and polarity. These two distinct properties of the amino acid side-chains jointly determine their preferred locations in folded proteins. The early appearance of an acceptor stem code based on size, β-branching, and carboxylate groups might have favored the appearance of antiparallel peptides that have been suggested to have a special affinity for RNA

Proton-in-Flight Mechanism for the Spontaneous Hydrolysis of N -Methyl O -Phenyl Sulfamate: Implications for the Design of Steroid Sulfatase Inhibitors

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 moderate efficiencies 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

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

Cytosine deamination appears to be largely responsible for spontaneous mutations in the modern world. Because of its sensitivity to temperature (Q10 = 4), that reaction would have furnished a mechanism for rapid evolution on a warm earth. As the temperature fell from 100° to 25 °C, the rate of cytosine-based mutation would have fallen by a factor of more than 4,000, with a corresponding increase in the stability of genetic information. Other potentially mutagenic events are known to be even more sensitive to temperature, and would presumably have led to an even steeper decline in the rate of spontaneous mutation as the earth cooled

Phosphate Monoester Hydrolysis in Cyclohexane

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 approximately 2 x 10(12)-fold by transfer from water to cyclohexane. That rate enhancement arises from an increase in the entropy of activation

Orotic Acid Decarboxylation in Water and Nonpolar Solvents: A Potential Role for Desolvation in the Action of OMP Decarboxylase

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 by 1-substituents that would allow it to enter less polar solvents. When the tetrabuytlyammonium 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 of similar 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

The hydrolysis of phosphate diesters in cyclohexane and acetone

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