The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Abstract

To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. A common property shared by enzymes catalyzing biological reactions that involve a single substrate and hydrolytic and hydration reactions in which the effective concentration of water (the second substrate) cannot be elevated much above its concentration in living tissue is their ability to lower the reaction heat of activation. The rates of these enzymatic reactions are less sensitive to temperature than are the rates of the uncatalyzed reactions, so that the rate enhancements and transition state affinities generated by enzymes of this kind increase with decreasing temperature. Entropic effects tend to be minor, with an average change in the entropy of activation that approaches zero (1). That behavior seems understandable in view of the absence of a second substrate or the fact that the second substrate (water) is present in abundance. Thus, approximation effects seem relatively unlikely to play a major role in catalysis by enzymes of those types. Instead, enthalpic effects play a prominent role in catalysis, consistent with the formation in the transition state of new electrostatic and H-bonds between the enzyme and substrate, for which much evidence exists in the structures of enzymes crystallized with transition state analogue inhibitors (1).The effects of enzymes on the enthalpies and entropies of activation of two-substrate reactions remain largely unexplored. The thermodynamics of activation (for an enzyme reaction and for the corresponding uncatalyzed reaction) were examined recently for an unusual two-substrate enzyme (2, 3). The peptidyl transferase center of the ribosome was found to produce a 3 ϫ 10 7 -fold enhancement of the rate of peptidyl ester aminolysis entirely by rendering T⌬S ‡ more favorable, whereas ⌬H ‡ was actually found to become less favorable on the enzyme than in solution (3). Juxtaposition, that is, the gathering of two or more substrates at an enzyme active site in a configuration appropriate for reaction, appears to be partly responsible for that rate enhancement, and desolvation of the substrates may also contribute to the observed rate enhancement (4, 5). Thus, the ribosome might be said to function as a predominantly "physical" catalyst, bringing the substrates near each other in a water-poor environment rather than as a predominantly "chemical" catalyst that participates in the reaction as a general acid or a general base. Consistent with that interpretation is the finding that ribosomal peptidyl transfer is very strongly inhibited by a bisubstrate analogue devised by Yarus and co-workers (6).Because of its unusual composition (consisting entirely of RNA) and the relatively small rate enhancement that it produces, the behavior of the peptidyltransferase center of the r...

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

“…This million-fold excess catalytic proficiency argues that Urzymes closely resemble true ancestral forms, that they are highly evolved, and hence had simpler functional ancestors. We show here that such ancestors might now themselves be experimentally accessible, especially in context of the literature on uncatalyzed rates (12)(13)(14)(15)(16), which both motivated this study and helped to shape the methods used.…”

“…(14). ATP hydrolyzes spontaneously over the same time frame (16) with a reported half-life of 212 days (0.0033 days Ϫ1 ). We confirmed experimentally that a similar rate (0.0035 days Ϫ1 ) applied to our assay conditions.…”

Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase

“…As in the case of other phosphate esters and phosphate anhydrides (21,22), each additional negative charge decreased the rate constant for PP i hydrolysis. Even at pH values as high as 13, ϳ4 pH units above the pK a value of PP i 3Ϫ , the observed rate constants reflect the hydrolysis of a small population of PP i 3Ϫ that reacts several orders of magnitude more rapidly than does the species predominating at pH 13, PP i 4Ϫ .…”

“…This behavior is consistent with the view that MgPP i 2Ϫ , rather than PP i 3Ϫ , is the major species that contributes to rate constants observed between pH 7.5 and 8.7. PP i Hydrolysis by Pyrophosphatase-As in earlier work on several kinase reactions (22), isothermal titration calorimetry furnished a sensitive continuous assay in experiments with E. coli PPase at pH 8.4. At this pH value, PP i hydrolysis is the slowest step, rather than substrate binding or product release (18).…”

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

“…Enthalpic effects have also been shown to predominate in systems in which catalysis is much less pronounced, including general base catalysis of the bromination of acetoacetate by glycolate (16), in the covalent catalysis of the hydrolysis of 4-nitrophenyl acetate by imidazole (17), in general base catalysis of the aminolysis of carboxylic esters by alkylamines (18), and in the Mg II -catalyzed methanolysis of ATP (19).…”