RESUMO
Kinetic analysis and protein mutagenesis allow the importance of individual amino acids in ligand binding and catalysis to be assessed. A kinetic analysis has shown that the reaction catalyzed by dihydrofolate reductase is optimized with respect to product flux, which in turn is predetermined by the active-site hydrophobic surface. Protein mutagenesis has revealed that specific hydrophobic residues contribute 2 to 5 kilocalories per mole to ligand binding and catalysis. The extent to which perturbations within this active-site ensemble may affect catalysis is discussed in terms of the constraints imposed by the energy surface for the reaction.
Assuntos
Mutação , Tetra-Hidrofolato Desidrogenase/metabolismo , Sítios de Ligação , Catálise , Fenômenos Químicos , Química , Escherichia coli/enzimologia , Cinética , Lacticaseibacillus casei/enzimologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/genética , TermodinâmicaRESUMO
The crystal structure of Bacillus subtilis ribonuclease P protein is reported at 2.6 angstroms resolution. This protein binds to ribonuclease P RNA to form a ribonucleoprotein holoenzyme with optimal catalytic activity. Mutagenesis and biochemical data indicate that an unusual left-handed betaalphabeta crossover connection and a large central cleft in the protein form conserved RNA binding sites; a metal binding loop may comprise a third RNA binding site. The unusual topology is partly shared with ribosomal protein S5 and the ribosomal translocase elongation factor G, which suggests evolution from a common RNA binding ancestor in the primordial translational apparatus.
Assuntos
Endorribonucleases/química , Evolução Molecular , Biossíntese de Proteínas , Conformação Proteica , RNA Bacteriano/química , RNA Catalítico/química , Bacillus subtilis/enzimologia , Sítios de Ligação , Catálise , Cristalografia por Raios X , Endorribonucleases/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Fator G para Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , RNA Bacteriano/metabolismo , RNA Catalítico/metabolismo , Ribonuclease P , Proteínas Ribossômicas/química , Zinco/metabolismoRESUMO
BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.
Assuntos
Aldeído Liases/química , Aldeído Liases/metabolismo , Domínio Catalítico , Mutação , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/enzimologia , Biblioteca Gênica , Cinética , Lisina/química , Modelos Químicos , Modelos Moleculares , Mutagênese , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
The ribonucleoprotein ribonuclease P catalyzes the hydrolysis of a specific phosphodiester bond in precursor tRNA to form the mature 5' end of tRNA. Recent studies have shed light on the structures of RNase-P-RNA-P-protein and RNase-P-RNA-precursor-tRNA complexes, as well as on the positions of catalytic metal ions, emphasizing the importance of the structure to the catalytic function.
Assuntos
Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , Sequência de Bases , Catálise , Endorribonucleases/química , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/metabolismo , Ribonuclease P , Especificidade por SubstratoRESUMO
Zinc metalloenzymes catalyze many important cellular reactions. Recently, the involvement of zinc in the catalysis of alkylation of sulfur groups has gained prominence. Current studies of the zinc metalloenzyme protein farnesyltransferase have shed light on its structure and catalytic mechanism, as well as the general mechanism of zinc-catalyzed sulfur alkylation.
Assuntos
Alquil e Aril Transferases/metabolismo , Enxofre/metabolismo , Zinco/metabolismo , Alquil e Aril Transferases/química , Alquilação , Catálise , Conformação ProteicaRESUMO
Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.
Assuntos
Anidrases Carbônicas/química , Proteínas/química , Aminoácidos/química , Isótopos de Carbono , Anidrases Carbônicas/genética , Deutério , Escherichia coli/genética , Humanos , Hidrogênio , Estrutura Molecular , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.
Assuntos
Anidrases Carbônicas/química , Dissulfetos/química , Dicroísmo Circular , Cristalografia por Raios X , Sistemas de Gerenciamento de Base de Dados , Humanos , Mutagênese , Engenharia de ProteínasRESUMO
[figure: see text] Linear free energy relationships between binding affinity and hydrophobicity for a library of fluoroaromatic inhibitors of F131V carbonic anhydrase II (CA) implicate three modes of interaction. X-ray crystal structures suggest that F131 interacts with fluoroaromatic inhibitors, while P202, on the opposite side of the active site cleft, serves as the site of the hydrophobic contact in the case of the F131V mutant. 2-Fluorinated compounds bind more tightly, perhaps due to the field effect of the nearby fluorine on the acidity of the amide proton.
Assuntos
Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Inibidores da Anidrase Carbônica/química , Cristalografia/métodos , Flúor/química , Transferência Linear de Energia , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
The inhibitor sensitivity of pulmonary vascular carbonic anhydrase (CA) was examined in situ to identify the specific isozyme responsible for vascular activity and to study its distribution in the lung. Vascular CA activity was monitored in isolated rat lungs by measuring the rate of CO2 excretion and the magnitude of postcapillary CO2-HCO(3-)-H+ disequilibria. Lungs were perfused with isotonic salines containing gluconate, sulfate, Cl-, or I-, with or without sulfonamide derivatives. Effects of a CA inhibitor purified from porcine blood plasma were also determined. Vascular CA activity was unaffected by gluconate, sulfate, Cl-, and I- (< or = 100 mM). Sulfonamides with vastly different rates of membrane permeation (i.e., readily permeating ethoxzolamide, slowly permeating acetazolamide, and membrane-impermeant quaternary ammonium sulfanilamide) were capable of accessing all vascular CA with similar rates of access. The porcine inhibitor of CA (340 nM) produced a significant, but submaximal, inhibition of vascular CA activity. The data suggest that pulmonary vascular activity reflects a high-activity membrane-bound isozyme, CA IV, which is located on the extracellular luminal surface of capillary endothelial cells.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Pulmão/enzimologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Anidrases Carbônicas/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Pulmão/efeitos dos fármacos , Masculino , Perfusão , Circulação Pulmonar/efeitos dos fármacos , Troca Gasosa Pulmonar/efeitos dos fármacos , Troca Gasosa Pulmonar/fisiologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.
Assuntos
Técnicas Biossensoriais/métodos , Anidrases Carbônicas/farmacologia , Hipocampo/metabolismo , Microscopia de Fluorescência/métodos , Zinco/metabolismo , Animais , Animais Recém-Nascidos , Anisotropia , Estimulação Elétrica , Humanos , Mamíferos , Técnicas de Cultura de Órgãos , Oxidiazóis/síntese química , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Sulfonamidas/síntese químicaAssuntos
Bioquímica/métodos , Enzimas/química , Simulação por Computador , Endorribonucleases/química , Endorribonucleases/metabolismo , Enzimas/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Análise de Injeção de Fluxo/métodos , Cinética , Modelos Químicos , RNA Catalítico/química , RNA Catalítico/metabolismo , Ribonuclease P , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
"Free Zn2+" (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or "tonic" level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5-25 nM). This concentration is 15,000-fold lower than the "300 microM" concentration which is often used as the "physiological" concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.
Assuntos
Anestésicos/metabolismo , Isquemia Encefálica/metabolismo , Sistema Nervoso Central/metabolismo , Espaço Extracelular/metabolismo , Reperfusão , Zinco/metabolismo , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Diálise/métodos , Eletroquímica/métodos , Espaço Extracelular/efeitos dos fármacos , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Coelhos , Ratos , Fatores de TempoRESUMO
Studies of the reactivity of succinyl-CoA:3-keto acid CoA transferase with a small coenzyme A analog, methylmercaptopropionate, have shown that noncovalent interactions between the enzyme and the side chain of CoA are responsible for a rate acceleration of approximately 10(12), which is close to the total rate acceleration brought about by the enzyme (Moore, S. A., and Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907). We report here that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of the rate acceleration and destabilization of the enzyme-thiol ester intermediate that is observed with CoA substrates. The role of the adenosine 3'-phosphate 5'-diphosphate moiety of CoA is to provide 6.9 kcal/mol of binding energy in order to pull the pantetheine moiety into the active site. The enzyme-thiol ester intermediate, E-pantetheine, was generated by reaction of pantetheine with the thiol ester of enzyme and methylmercaptopropionate. E-Pantetheine undergoes hydrolysis with khyd = 2 min-1, 140-fold faster than E-CoA, and reacts with acetoacetate with kAcAc = 3 X 10(6) M-1 min-1, only 10-fold slower than E-CoA. However, in the reverse direction acetoacetylpantetheine reacts with CoA transferase (kAcAc-SP = 220 M-1 min-1) 1.6 X 10(6) times slower than acetoacetyl-CoA. The equilibrium constant for the reaction of pantetheine with E-CoA is approximately 8 X 10(-6).
Assuntos
Difosfato de Adenosina/análogos & derivados , Coenzima A-Transferases , Coenzima A/metabolismo , Panteteína/farmacologia , Compostos de Sulfidrila/farmacologia , Sulfurtransferases/metabolismo , Acetoacetatos/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Ativação Enzimática , Cinética , Miocárdio/enzimologia , Propionatos/farmacologia , Ligação Proteica , SuínosRESUMO
Carbonic anhydrase II (CAII) contains a conserved His3 zinc polyhedron which is essential for catalysis. Removal of any one of the His ligands by replacement with Ala decreases (approximately 10(5)-fold), but does not abolish, zinc binding and increases the rate constant for zinc dissociation. CAII variants with a His ligand substituted with Cys, Asp, or Glu bind zinc only approximately 10-fold better than a His2 zinc polyhedron in CAII. The large decrease in zinc affinity (approximately 5 kcal/mol) in these variants compared to the wild-type His3 site reflects mainly unfavorable compensatory protein structural rearrangements observed in the X-ray crystallographic structures of some of these CAII variants, described by Ippolito and Christianson (following paper in this issue). However, the zinc affinity of these sites is still higher than zinc polyhedra designed de novo. Substitution of the His zinc ligands with negatively charged amino acids both increases the pKa of the zinc-bound water by > or = 1.6 pH units, confirming that neutral ligands maintain the low zinc-water pKa, and decreases the pH-independent kcat/KM for ester hydrolysis (3-30-fold) and CO2 hydration (approximately 10(3)-10(5)-fold). Additionally, decreases in the dissociation constant (approximately approximately 10(2)-10(5)-fold) for the transition state analog acetazolamide correlate with the decreased catalytic efficiency and increased pKa of these CAII variants. These data indicate that the histidine ligands, although not essential for catalysis, are conserved to maximize electrostatic stabilization of both the ground-state zinc-hydroxide and the negatively charged transition state. These studies provide valuable insights into the functional consequences of engineering a catalytic zinc site in a metalloenzyme.
Assuntos
Anidrases Carbônicas/química , Sítios de Ligação , Dióxido de Carbono/química , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Metaloproteínas/química , Mutagênese Sítio-Dirigida , Solventes , Relação Estrutura-Atividade , Água/química , Zinco/químicaRESUMO
A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees C). This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques. A minimal kinetic description involves the following: (1) binding of pre-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M-1 s-1), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-1; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (> or = 0.2 s-1) followed by rate-limiting tRNA dissociation (0.02 s-1); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 x 10(4) M-1 s-1). This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions. This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity.
Assuntos
Bacillus subtilis/enzimologia , Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Sítios de Ligação , Endorribonucleases/química , Hidrólise , Cinética , Estrutura Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Catalítico/química , RNA de Transferência de Ácido Aspártico/química , Ribonuclease P , TermodinâmicaRESUMO
Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with Ki < 0.1 nM for porcine CA II at pH 7.4. Although the Ki is dependent on the CA isozyme type (CA II << CA IV << CA III approximately CA I), it is relatively insensitive to the species source, as long as it is mammalian. The Ki is pH dependent with log Ki decreasing linearly as the pH decreases, implicating at least one ionizable group with the pKa < or = 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.
Assuntos
Inibidores da Anidrase Carbônica/isolamento & purificação , Animais , Inibidores da Anidrase Carbônica/sangue , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Peso Molecular , Conformação Proteica , Especificidade da Espécie , Especificidade por Substrato , SuínosRESUMO
Measurement of free zinc levels and imaging of zinc fluxes remains technically difficult due to low levels and the presence of interfering cations such as Mg and Ca. We have developed a series of fluorescent zinc indicators based on the superb sensitivity and selectivity of a protein, human apo-carbonic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotropy; the latter three approaches permit quantitative imaging of zinc levels in the microscope. A unique attribute of sensors incorporating biological macromolecules as transducers is their capability for modification by site-directed mutagenesis. Thus we have produced variants of carbonic anhydrase with improved affinity for zinc, altered selectivity, and enhanced binding kinetics, all of which are difficult to modify in small molecule indicators.
Assuntos
Técnicas Biossensoriais , Anidrases Carbônicas/química , Corantes Fluorescentes , Zinco/análise , Animais , Sítios de Ligação , Química Encefálica , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Engenharia de ProteínasRESUMO
The role of Thr-113 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Thr-113, a strictly conserved residue that forms a hydrogen bond to the active-site Asp-27 and to the amino group of methotrexate through a fixed water molecule, was replaced by valine. The kinetic scheme is identical in form with the wild-type scheme, although many of the rate constants vary, including a decrease in the association rate constants and an increase in the dissociation rate constants for folate ligands, a decrease in the hydride-transfer rate constant in both directions, and an increase in the intrinsic pKa of Asp-27. Overall, replacement of Thr-113 by Val decreases the binding of folate substrates by approximately 2.3 kcal/mol. These multiple complex changes on various ground and transition states underscore the optimal properties of a strictly conserved residue in the evolution of catalytic function.