Kinetic Consequences Research Articles

The Type 2 Intramolecular Diels–Alder Reaction: Synthesis and Chemistry of Bridgehead Alkenes

Anti-Bredt alkenes, bicyclic molecules that contain a bridgehead double bond, were for many years regarded as chemical curiosities. The type 2 intramolecular Diels–Alder (IMDA) reaction provides a one-step entry into this fascinating class of molecules. The reaction has made available numerous anti-Bredt alkenes for structural and chemical studies. X-ray crystallography has revealed the magnitude of the deformations associated with the bridgehead double bond, and rate studies of reactions of bridgehead alkenes have allowed quantification of the kinetic consequences of the torsional distortions. More recently, the type 2 intramolecular Diels–Alder reaction and the resulting anti-Bredt alkenes have found application in organic synthesis. The constraints resulting from the connectivity in the Diels–Alder precursor creates a strong regio- and stereochemical bias in the cycloaddition step. The end result of this bias is the stereoselective synthesis of highly substituted six-membered rings. The reaction also achieves a facile synthesis of seven- and eight-membered rings in a single step from acyclic precursors. The utility of this reaction has been verified in recent applications of the type 2 IMDA reaction as a key step in the total synthesis of complex natural products.

Structural, thermodynamic and kinetic consequences of a spectroscopic study of the equilibrium between isomeric forms of ferrocene-containing β-diketones

Proton NMR showed that, at equilibrium and at 25°C, asymmetric enolization in the direction furthest from the ferrocenyl group is dominant for the β-diketones 1-ferrocenyl-4,4,4-trifluoro-1,3-butanedione (ferrocenoyltrifluoroacetone, Hfctfa), 1-ferrocenyl-4,4,4-trichloro-1,3-butanedione (ferrocenoyltrichloroacetone, Hfctca), 1-ferrocenyl-1,3-butanedione (ferrocenoylacetone, Hfca), 1,3-diferrocenyl-1,3-propanedione (diferrocenoylmethane, Hdfcm) and 1-ferrocenyl-3-phenyl-1,3-propanedione (benzoylferrocenoylmethane, Hbfcm). This finding is considered to be the result of resonance driving forces rather than inductive electronic effects of substituents on the pseudo-aromatic β-diketone core. Lowering of the concentration of the β-diketones from 35 to below 1 mmol dm −3 only slightly shifts the equilibrium more towards the keto side. By increasing the temperature of the solvent (CDCl 3) from 20 to 60°C, the percentage keto isomer at equilibrium of Hdfcm (32.9–34.2%), Hfca (22.5–28.4%), Hbfcm (8.8–11.9%) and Hfctca (4.9–9.8%) increased but it decreased the keto percentage of Hfctfa from 3.2 to below 0.7%. Slow conversion kinetics from the keto to the dominant enol isomer explains why, directly after isolation of newly prepared compounds, higher percentages of the keto isomer are observed.

The Type 2 Intramolecular Diels-Alder Reaction: Synthesis and Chemistry of Bridgehead Alkenes

Anti-Bredt alkenes, bicyclic molecules that contain a bridgehead double bond, were for many years regarded as chemical curiosities. The type 2 intramolecular Diels-Alder (IMDA) reaction provides a one-step entry into this fascinating class of molecules. The reaction has made available numerous anti-Bredt alkenes for structural and chemical studies. X-ray crystallography has revealed the magnitude of the deformations associated with the bridgehead double bond, and rate studies of reactions of bridgehead alkenes have allowed quantification of the kinetic consequences of the torsional distortions. More recently, the type 2 intramolecular Diels-Alder reaction and the resulting anti-Bredt alkenes have found application in organic synthesis. The constraints resulting from the connectivity in the Diels-Alder precursor creates a strong regio- and stereochemical bias in the cycloaddition step. The end result of this bias is the stereoselective synthesis of highly substituted six-membered rings. The reaction also achieves a facile synthesis of seven- and eight-membered rings in a single step from acyclic precursors. The utility of this reaction has been verified in recent applications of the type 2 IMDA reaction as a key step in the total synthesis of complex natural products.

Kinetic consequences of covalent linkage of DNA binding polyamides.

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.

Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.

The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of these observations a revised mechanism for fructose-2,6-bisphosphatase that is consistent with all of the previously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences produced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAMP-dependent phosphorylation of Ser-32, which is located in the N-terminal kinase domain.

Spectroscopy and relaxation kinetics of the perturbed CO(b 3Σ+,v′=0,1,2) and CO(a′ 3Σ+, v′=31–36, 40, and 41) levels and reinterpretation of CO(a′ 3Σ+, v′=34 and 35) formation in the Kr(5s′[1/2])+CO reaction

The spectroscopic and kinetic consequences associated with the strong homogeneous interactions between (i) CO(b 3Σ+,v′=0) and CO(a′ 3Σ+, v′=30, 31, and 32), (ii) CO(b 3Σ+,v′=1) and CO(a′ 3Σ+, v′=34, 35, and 36), (iii) CO(b 3Σ+,v′=2) and CO(a′ 3Σ+, v′=40 and 41) levels are evaluated. Mixing of b 3Σ+ character into the a′ 3Σ+ levels reduces the radiative lifetimes of the latter, because the lifetimes of pure b 3Σ+ and a′ 3Σ+ states are approximately 50 ns and 3 μs, respectively. The strength of the interaction changes with rotational level, and the rotational line intensities and the radiative branching to different v″ levels of the CO(a′ 3Σ+–a 3Π) transition are strongly affected. Comparison of high resolution CO(a′, v′=31, 34, and 35) experimental spectra with calculated spectra shows a marked underestimation of the rotational temperature of these CO(a′,v′) levels unless the mixing is explicitly recognized. With benefit of this knowledge, some results from the Kr(5s′[1/2]0)+CO excitation-transfer reaction need to be reinterpreted. Emission spectra for 300 K rotational distributions from CO(b,v′=0, 1, and 2) and CO(a′,v′=30, 31, 34, and 35) were used to obtain vibrational band intensities for comparison with model calculations. Analysis of the pressure and time dependence of the laser-induced fluorescence data permit the electronic relaxation mechanism of the CO(b,v′=0, 1, 2) and CO(a′,v′=31 and 35) levels in He buffer gas to be discussed. The experimental radiative lifetimes of CO(b,v′=0,1,2) were measured as 60±6, 63±4, and 58±4 ns, respectively. The role of the homogeneously perturbed levels in the collisional relaxation mechanism is discussed.

Fundamental reactions of free radicals relevant to pyrolysis reactions

Free-radical mechanisms are ubiquitous for pyrolysis of organic substrates. Background information is supplied to assist in the interpretation of such reactions by dissection into the elementary radical-forming, radical-consuming, and radical-interconverting reactions that form the building blocks of overall mechanisms. Particular attention is given to homolysis, hydrogen abstraction, β-scission–addition, and rearrangement steps, and to bond-breaking and bond-forming processes. Since the thermochemistry of such elementary reactions is dependent on the stability of the radicals involved, the major influences of structural changes on radical stability are reviewed. Kinetics of prototypical elementary reactions and their responses to structural changes are rationalized in terms of their Arrhenius parameters. The fundamental relationships between thermochemical parameters and kinetic parameters are summarized, and the use of these to estimate rate constants for cases where data is not available is demonstrated. Some kinetic consequences of the combination of elementary reactions to produce chain reactions are reviewed. Critical structural and kinetic features that determine the selectivity among product types and the response of reactions to temperature and substrate concentration are indicated.

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P 1 and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 Å at room temperature allowed anisotropic structure refinement characterized by R = 0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C α atoms of 0.7 Å at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO 4 2− anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular β-sheet that, together with the adjacent intramolecular β-hairpin loops, creates a four-stranded structure.

Increased catalytic performance of the 2-oxoacid dehydrogenase complexes in the presence of thioredoxin, a thiol–disulfide oxidoreductase

Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase complexes undergo an irreversible inactivation upon accumulation of the dihydrolipoate intermediate. The first component of the complexes, 2-oxoacid dehydrogenase, is affected. Addition of thioredoxin protects from this inactivation, increasing catalytic rates and limiting degrees of the substrate transformation to products, acyl-CoA and NADH. Although the redox active cysteines of thioredoxin are essential for its interplay with the complexes, the effects are observed with both dithiol and disulfide forms of the protein. This indicates that thioredoxin affects an SH/S–S component of the system, which is present in the two redox states. The complex-bound lipoate is concluded to be the thioredoxin target, since (i) both dithiol and disulfide forms of the residue are available during the catalytic cycle and (ii) the thioredoxin reaction with the essential SH/S–S group of the terminal component of the complex, dihydrolipoyl dehydrogenase, is excluded. Thus, the thioredoxin disulfide interacts with the dihydrolipoate intermediate, while the thioredoxin dithiol reacts with the lipoate disulfide. Kinetic consequences of such interplay are consistent with the observed thioredoxin effects. Owing to the essential reactivity of the SH/S–S couple in thioredoxin, the thiol–disulfide exchange between thioredoxin and the lipoate residue is easy reversible, providing both protection (by the mixed disulfide formation) and catalysis (by the appropriate lipoate release). In contrast, non-protein SH/S–S compounds prevent the inactivatory action of dihydrolipoate intermediate only at a high excess over the complex-bound lipoate. This interferes with the catalysis-required release of the residue from its mixed disulfide. Therefore, only thioredoxin is capable to `buffer' the steady-state concentration of the reactive dithiol. Such action represents a new thioredoxin function, which may be exploited to protect other enzymes with exposed redox-active thiol intermediates.

Resolving the Control of Magnesium on Calcite Growth: Thermodynamic and Kinetic Consequences of Impurity Incorporation for Biomineral Formation

ABSTRACTMagnesium is a key determinant in CaCO3 biomineral formation and has recently emerged as an important paleotemperature proxy. Atomic force microscopy (AFM) was used to determine the fundamental thermodynamic and kinetic controls of Mg2+ on calcite morphology and growth. Comparison of directly measured monomolecular step velocities (vs±) to theoretical crystal growth impurity models demonstrated calcite inhibition due to enhanced mineral solubility through Mg2+ incorporation. Terrace width (λ) measurements independently supported an incorporation mechanism by indicating a shift in the effective supersaturation (σeff) of the growth solutions in the presence of Mg2+. This study resolves the controversy over the molecular-scale mechanism of calcite inhibition by Mg2+ and provides an unambiguous model for the thermodynamic and kinetic consequences of impurity incorporation into CaCO3 biominerals.

Syntheses of imidazolium‐bridged cyclodextrin dimers and their catalytic properties in the hydrolytic cleavage of p‐nitrophenyl alkanoates

AbstractTwo imidazolium‐bridged cyclodextrin dimers 3a and 3b were prepared by reacting 6‐deoxy‐6‐N‐imidazolyl‐β‐CD (2) with bis(bromomethyl) benzene. The catalytic properties of 2, 3a and 3b in the hydrolytic cleavage of p‐nitrophenyl alkanoates, in the form of acetate (PNPA), butanoate (PNPB), hexanoate (WH) and octanoate ( PNPO), were examined. CD dimers showed middling rate enhancements around neutrality. Catalytic rate constants ( kc) in the presence of 3a or 3b did not vary much with chain length of esters. In contrast, dissociation constants (Kd) and selectivity factors (kc/Kd) for “long‐chain” esters were much smaller and significantly larger than those for “short‐chain” ones respectively, indicating CD dimers 3a and 3b have good dimensional recognition ability and substrate selectivity in the hydrolytic cleavage of p‐nitrophenyl alkanoate. Their kinetic consequences are briefly interpreted.

Disturbed Communication between Actin- and Nucleotide-binding Sites in a Myosin II with Truncated 50/20-kDa Junction

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.

How locomotor parameters adapt to gravity and body structure changes during gait development in children.

The principles of direct dynamics and oscillating systems have been used to study the development of gait parameters in children, with respect to their kinetic consequences on the oscillations of body center of mass (CM). In particular the equations established (a) a natural body frequency (NBF), a body parameter specific to oscillating movements which is invariant for adults and decreases with age for children, and (b) the amplitude ratio of CM to center-of-foot pressure (CP) oscillations as a parametric function of the step frequency, whose parameter is the NBF. This function was used to analyze the development of gait locomotors with respect to their kinetic effects on balance in the frontal plane. Five children were examined longitudinally during their first 5 years of independent walking (IW), and two cross-sectional groups between 5 and 7 years of IW were also considered. The results showed a shift toward the low end of step frequency bands as the NBF decreased along with invariances in the amplitudes of CM oscillation in both the frontal and sagittal planes, regardless of age and gait velocity. The biomechanical meaning of the NBF, of its decrease and of postural invariances associated with the decrease of the frequency, are discussed as well as how the programming of locomotor parameters adapts to changes in body structure during gait development.

On the Effect of Ocean Waves on the Kinetic Energy Balance and Consequences for the Inertial Dissipation Technique

Abstract For large wind speed (in practice >15 m s−1) observations of the surface stress by means of the inertial dissipation technique are so close to the surface that effects of growing ocean waves on the turbulent kinetic energy budget should be taken into account. This may give rise to an increase in the surface stress of 20%.

安定中間体効果と寄生平衡 光誘発反応を制御する重要な速度論的因子

This review deals with two important kinetic effects based on concentration changes of radical and radical ion intermediates, which in some cases dominate the efficiency and selectivity of photoinduced electron transfer (PET) reactions. One is the persistent radical ion effect suggesting the predominant reaction of a more stable radical ion rather than that of less stable, i. e., more reactive, intermediate, just as the Ingold-Fischer persistent radical one which predicts high yields of the cross-reaction product from two radicals with markedly different self-termination constants. In PET reactions between D and A, reactions under continuous irradiations are in principle governed by the steady-state concentrations of radical ion intermediates, the ratios of D+· and A-· being quite different from 1 : 1 depending on their relative stabilities. The kinetics are more simple and the ratio of D+· and A-·becomes 1 : 1 when intermediates are generated by pulsed irradiation. Such a contrast was demonstrated for the redox between diphenylmethyl radical and simultaneously generated aromatic radical cation/anion, and for the photosensitized redox reaction of carboxylates. Another important effect is based on abortive equilibria, indicating that a reactive intermediate exists in an equilibrium with stable species not leading to products. Kinetic consequences of this effect are described for the deprotonation of diarylmethane radical cations with pyridines, and for the PET oxidative C-C cleavage of 1, 1, 2, 2-tetraphenylethane.

Kinetic Model of Folate Metabolism in Nonpregnant Women Consuming [2H2]Folic Acid: Isotopic Labeling of Urinary Folate and the Catabolite para-Acetamidobenzoylglutamate Indicates Slow, Intake-Dependent, Turnover of Folate Pools

In a 10-wk study of folate metabolism in nonpregnant women (21–27 y, n –6 per group), subjects were fed a diet containing ∼68 nmol/d (30 μg/d) folate from food. The remainder of the ingested folate was provided as folic acid in apple juice (as nonlabeled during wk 1–2, as [2H2]folic acid during wk 3–10) to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 μg/d). Isotopic enrichment of total urinary folate and the primary catabolite para-acetamidobenzoylglutamate (ApABG) was determined. Isotopic enrichment of ApABG served as an indicator of labeling of tissue folates. A kinetic model consisting of fast- and slow-turnover nonsaturable pools and a saturable slow-turnover pool, with provisions for urinary and fecal excretion, catabolism and enterohepatic circulation, yielded a close fit to the data. Mean residence times for total body folate were 212, 169 and 124 d for folate intakes of 454, 680, and 907 nmol/d, respectively. The model predicted that variation in folate intake over this range had little effect on the mass of the large saturable folate pool; however, the fast-turnover nonsaturable pools increased in proportion to folate intake, whereas the slow nonsaturable pool also tended to increase. This model will aid in evaluation of folate turnover and in predicting kinetic consequences of physiologic conditions associated with altered folate requirements.

Surface smoothing during sputtering: mobile vacancies versus adatom detachment and diffusion

Surface smoothing during sputtering can occur through both vacancy diffusion and adatom diffusion. In this paper, using kinetic Monte Carlo simulations, we investigate the kinetic consequences of smoothing with either mechanism acting alone. Quasi-layer-by-layer sputtering and an approximately Arrhenius variation of pit density during submonolayer sputtering are observed with both types of surface smoothing. However, the two mechanisms result in significant differences in the pit size distribution for submonolayer sputtering and the vacancy coverage at which the maxima of the anti-Bragg specular beam intensity oscillations occur during layer-by-layer sputtering. These and other experimentally observable quantities are investigated as possible ways to determine the relative importance of the two smoothing mechanisms.

The RNA folding problem: a variational problem within an adiabatic approximation

Biopolymer folding is an expeditious process taking place within timescales incommensurably shorter than ergodic times. Furthermore, its robustness suggests that the process must depend on a relatively coarse level of resolution of conformation space. To account for these features while focusing on the RNA context, we derive a variational principle formulated within an adiabatic approximation obtained by integrating out fast-relaxing molecular motions. Folding pathways are generated by means of a stochastic process which begets a least effort principle reflecting a stepwise minimization of the conformational entropy cost for each folding event with concurrent maximization of the base pairing. This economy of the process is found to have kinetic consequences if we treat base-pairing contact patterns (BPPs) adiabatically, that is, as quasi-equilibrium states: the probability distribution of overall folding timespans associated to the process resolved at the BPP level is maximized at the brachistochrone or overall least-time pathway for functionally-competent RNAs. In turn, this pathway is shown to yield all the phylogenetically-conserved structural features of the active conformation within biologically-relevant timescales.

Kinetic mechanism of UDP-hexose synthase, a point variant of hexose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate (galactose-1-P) uridylyltransferase from Escherichia coli catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism through a uridylyl-enzyme intermediate, in which the uridine-5'-phosphoryl group is covalently bonded to Nepsilon of His 166. The point variant H166G displays a UDP-hexose synthase activity, in that it catalyzes the reaction of uridine 5'-phosphoimidazolide (UMPIm) with glucose-1-P to form UDP-glucose and imidazole. Inasmuch as the wild-type uridylyltransferase catalyzes its cognate reaction with ping-pong kinetics, an intrinsically ordered substrate binding mechanism, the kinetic mechanism of the UDP-hexose synthase activity of H166G became of interest. The synthase activity follows sequential kinetics [Kim, J., Ruzicka, F., and Frey, P. A. (1990) Biochemistry 29, 10590-10593]. In this work, product inhibition patterns for the synthase activity of H166G indicate random equilibrium binding of substrates. Comparison of the synthase activities of the variants H166G and H166A showed that the glycine variant is about 340- and 600-fold more active than the alanine variant in the forward and reverse directions, respectively. The kinetic consequences of varying the amino acid at position 166 were largely kcat effects, with more modest Km effects. Comparison of the synthase activities of these variants with that of the wild-type enzyme in the production of glucose-1-P showed that the loss of the beta-carbon of His 166 in the complex H166G-UMPIm increases the activation energy for uridylyl group transfer by 2.4 kcal mol-1, and the presence of two additional hydrogen atoms in the complex H166A-UMPIm increases the activation energy by 6.2 kcal mol-1. It is concluded that the active site is much less tolerant of additional steric bulk in the locus of the beta-carbon of His 166 than it is of the loss of the beta-carbon. The sensitivities to additional steric bulk around other positions of the His 166-imidazole ring are much less severe, as indicated by the reactivities of methylated analogues of UMPIm in the synthase reaction of H166G. Uridine 5'-phospho-N-methylimidazolide is more reactive as a synthase substrate than UMPIm, and this is attributed to the positive charge of the imidazole ring. The fact that the imidazole ring of the wild-type covalent uridylyl-enzyme retains its proton and is positively charged is supported by the pH-rate profile for hydrolysis of the intermediate.

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.