The rate enhancement produced by the ribosome: an improved model

As a model for mechanistic comparison with peptidyl transfer within the ribosome, the reaction of aqueous glycinamide with N-formylphenylalanine trifluoroethyl ester (fPhe-TFE) represents an improvement over earlier model reactions involving Tris. The acidity of trifluoroethanol (pKa 12.4) resembles that of tRNA (12.98) more closely than do the acidities of model reactants described earlier, and the reactivity of the simple nucleophile glycinamide is free of potential complications that arise from alternative reaction pathways available to Tris. At 25 degrees C, the uncatalyzed reaction of glycinamide with fPhe-TFE proceeds with a second-order rate constant of 3 x 10(-5) M-1 s-1; DeltaH(++) = +7.8 kcal/mol; TDeltaS(++)= -15.7 kcal/mol. The ribosomal reaction of puromycin with fMet-tRNA proceeds 3 x 107-fold more rapidly, with a second-order rate constant (kcat/Km) of 1 x 10(3) M-1 s-1; DeltaH(++) = +16.0 kcal/mol; TDeltaS(++)= +2.0 kcal/mol. That rate enhancement, an order of magnitude larger than estimated earlier, is fully explained by the more favorable entropy of activation of the ribosomal reaction. Experiments involving ethylene glycol esters suggest that neighboring -OH group effects are negligible in the presence of solvent water, which itself acts as a general base catalyst. In the desolvated interior of the ribosome, the vicinal 2'-OH group of aminoacyl-tRNA probably replaces water as a general base catalyst. But the catalytic effect of the ribosome itself is overwhelmingly entropic in origin, suggesting that the ribosome achieves its effect by physical desolvation and/or juxtaposition of the reactants in a manner conducive to peptidyl transfer.     

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (DeltaH(++) of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298 K. Phosphate transfer from Cdk2-pTpY/CycA to enzyme (DeltaH(++) of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (DeltaH(++) of 18 kcal/mol), yet entropically less favorable (TDeltaS(++) of 2 vs. -6 kcal/mol at 298 K, respectively). By measuring the temperature dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy-entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme-substrate association involves tight and specific contacts at the remote docking site and that phospho-transfer from Cdk2-pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.     

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.     

Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Redefining the minimal substrate tolerance of mandelate racemase. Racemization of trifluorolactate

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the enantiomers of mandelic acid and a variety of aryl- and heteroaryl-substituted mandelate derivatives, suggesting that β,γ-unsaturation is a requisite feature of substrates for the enzyme. We show that β,γ-unsaturation is not an absolute requirement for catalysis and that mandelate racemase can bind and catalyze the racemization of (S)-trifluorolactate (k(cat) = 2.5 ± 0.3 s(-1), K(m) = 1.74 ± 0.08 mM) and (R)-trifluorolactate (k(cat) = 2.0 ± 0.2 s(-1), K(m) = 1.2 ± 0.2 mM). The enzyme was shown to catalyze hydrogen-deuterium exchange at the α-postion of trifluorolactate using (1)H NMR spectrocsopy. β-Elimination of fluoride was not detected using (19)F NMR spectroscopy. Although mandelate racemase bound trifluorolactate with an affinity similar to that exhibited for mandelate, the turnover numbers (k(cat)) were markedly reduced by ～318-fold, resulting in catalytic efficiencies (k(cat)/K(m)) that were ~400-fold lower than those observed for mandelate. These observations suggested that chemical steps on the enzyme were likely rate-determining, which was confirmed by demonstrating that the rates of mandelate racemase-catalyzed racemization of (S)-trifluorolactate were not dependent upon the solvent microviscosity. Circular dichroism spectroscopy was used to measure the rates of nonenzymatic racemization of (S)-trifluorolactate at elevated temperatures. The values of ΔH(?) and ΔS(?) for the nonenzymatic racemization reaction were determined to be 28.0 (±0.7) kcal/mol and -15.7 (±1.7) cal K(-1) mol(-1), respectively, corresponding to a free energy of activation equal to 33 (±4) kcal/mol at 25 °C. Hence, mandelate racemase stabilizes the altered trifluorolactate in the transition state (ΔG(tx)) by at least 20 kcal/mol.     

A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.     

A calorimetric study of 3d metal ions-acyclovir interactions. The 2-hydroxyethoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting the coordination through N(7)

The equilibrium constants and enthalpic values of metal acyclovir complexes have been determined by calorimetry for Co(II) (log K=0.96+/-0.05, DeltaH (kJ/mol)=-19.7+/-1.3), Ni(II) (log K=1.39+/-0.03, DeltaH (kJ/mol)=-21.5+/-1.0), Cu(II) (log K=1.83+/-0.03, DeltaH (kJ/mol)=-23.2+/-0.8) and Zn(II) (log K=0.71+/-0.06, DeltaH (kJ/mol)=-18.6+/-1.5). The equilibrium constants are similar to those of the divalent ions with guanosine and 2,9-dimethylpurine. By comparison with previous thermodynamic data, it can be shown that the 2-hydroxyethoxymethyl group promotes coordination through N(7) versus N(1) of the guanine ring for 3d metal ions. These results reveal that the 2-hydroxyethoxymethyl group placed on the purine ring of guanine in acyclovir causes a greater effect than that of the 9-methyl in purines and similar to or greater than that of the ribose moiety in guanosine. The 2-hydroxyethyoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting a similar coordination chemistry (with very close log K and DeltaH values) for acyclovir, deoxy-guanosine and guanosine with divalent metals.     

The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes. A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5-0.8 mol molybdenum, 0.6-0.9 mol pterin, 28 +/- 2 mol non-heme iron and 28 +/- 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 mumol.min-1.mg-1 (kcat = 640 s-1) assayed with methylviologen (app. Km = 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.     

Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.     

Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.     

Purification and characterization of rat liver transglutaminase

1. Transglutaminase (EC 2.3.2.13) was purified from rat liver. 2. The enzyme was stable at 25 degrees C in the pH range of 6.0-9.0, with the optimum at pH 9.0. 3. The enzyme was inactivated after incubation for 20, 4 and 1 min at 44 degrees C, 52 degrees C, and 60 degrees C, respectively. 4. Activation energies were 30.4 kcal/mol for denaturation and 19.9 kcal/mol for substrate conversion to products. 5. The enzyme was inactivated by sulfhydryl modification with hydroxymercuribenzoate (99.1%) and N-ethylmalemide (78.5%). 6. Calcium, required for the activity, was replaced to a lesser extent, by Mg2+, Sr2+, Zn2+ and Mn2+ (31.8, 27.0, 24.6 and 3.5%). 7. Steady-state kinetics showed: Vmax = 10 microM-min-1, Km = 0.05 mM (N-dimethylated casein), kcat = 31.9 min-1 kcat/Km = 560 min-1 mM-1.     

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.