ES Complex Research Articles

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pKa = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pKa = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pKa = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined Kd values for the ES complex formed with L-proline agree with results obtained in spectral titrations with the wild-type or mutant enzyme. Unlike the wild-type enzyme, Kd values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pKa of a group that affects the Kd. As compared with the wild-type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogues while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with the L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pKa, reaction rates, and charge transfer bands.

Purification and characterization of caprine kidney uricase, possessing novel kinetic and thermodynamic properties

Purified uricase from a caprine kidney, possessed Km and Vmax values of 1.1 mg ml−1 and 3512 IU (mg protein)−1 for uric acid hydrolysis, respectively. The optimum temperature and pH for catalytic activity were 40 °C and 8.5, respectively. The activation energy for formation of ES complex was 13.6 kJ mol−1. Enthalpy (ΔH*), entropy of activation (ΔS*) and Gibbs free energy demand of uricase inactivation were 62.8 kJ mol−1, −102 J mol−1 K−1 and 104.3 kJ mol−1, respectively. Gibbs free enrgy demand for substrate binding and transition state stabilization were also determined which were comparable with those for themostable enzymes.

The Fe(III)Zn(II) form of recombinant human purple acid phosphatase is not activated by proteolysis

The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe 3+Fe 2+ human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in p K es,1 (the value of an acid dissociation constant of the ES complex). These differences are due to the presence or absence, respectively, of an interaction between an aspartate residue in an exposed loop of the protein and one or more active site residues. To further explore the origin of these differences, the ferrous ion of recHPAP has been replaced by zinc. Analysis of the reconstituted Fe 3+Zn 2+recHPAP reveals an unexpected catalytic activity versus pH profile, in that the optimal pH is 6.3, similar to that of the proteolytically cleaved form (6.5). Moreover, replacement of the ferrous ion by zinc increases the turnover number more than 10-fold; the p K es values are also shifted as expected for the change in the divalent metal ion. Although the EPR spectra of both single polypeptide and proteolytically cleaved Fe 3+Zn 2+-recHPAP are independent of pH over the range 4.5–6.2, the visible spectrum of Fe 3+Zn 2+-recHPAP is pH dependent. These results suggest that the properties and environment of the divalent metal are important in determining the catalytic properties of mammalian PAPs, and in particular that a solvent molecule coordinated to the divalent metal ion may play a critical role in the catalytic cycle of these enzymes.

The Effect of Cations on the Amidase Activity of Human Tissue Kallikrein: 1-Linear Competitive Inhibition by Sodium, Potassium, Calcium and Magnesium. 2-Linear Mixed Inhibition by Aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.

Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

The α2β2 tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the α-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the α- and β-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1apo), the holoenzyme (E1holo), E1holo in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1holo in complex with 4-methyl-2-oxopentanoate (MOPA) as the α-carbanion/enamine intermediate (E1ceim). Binding of cofactors to E1apo induces a disorder–order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1holo, the loop comprised of Gly121β-Gln131β moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86β and N4′ of ThDP. The hydrophobic moiety of MPA is recognized by Phe66α, Tyr95α, Met128α and His131α. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open–closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1holo·MOPA and of E1ceim·lipoamide built from the above structures suggest that His273α and His129β′ are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.

Phosphatidylinositol-Specific Phospholipase C Forms Different Complexes with Monodisperse and Micellar Phosphatidylcholine

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex. The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase. The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex.

Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy.

The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2'-deoxy-FAD. The k(cat) (18.8 +/- 0.5 s(-1)) and K(m) (2.5 +/- 0.4 microM) values for the oxidation of n-octanoyl-CoA (C(8)-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257-267], reconstitution of the WT enzyme with 2'-deoxy-FAD causes a large (400-fold) decrease in k(cat) but has little effect on K(m). To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2'-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm(-1) decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm(-1), the enone band is only shifted 10 cm(-1) upon binding HD-CoA to 2'-deoxy-FAD pMCAD. Thus, removal of the 2'-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol(-1), while the activation energy for the reaction is raised 15 kJ mol(-1). In addition, removal of the 2'-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol(-1). Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C(8)-CoA alpha-protons with solvent deuterium, the 2'-deoxy-FAD-reconstituted enzyme catalyzes alpha-proton exchange at a rate (k(exc)) of 0.085 s(-1), which is only 4-fold slower than k(exc) for WT pMCAD (0.35 s(-1)). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for alpha-proton exchange, in contrast to its role in substrate oxidation, or the value of k(exc) for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.

Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var. mycoides cocrystallized with maltopentaose.

The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state.

Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg(40) and Arg(42) on small loop 1, a cluster Lys(193)-Lys(195) on small loop 2, and two sites, Arg(94) and Arg(118)-Lys(119), on the large loop were identified as binding sites. Lys(87) on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg(94) and Lys(193)-Lys(195) located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg(118)-Lys(119) was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

Kinetic Peculiarities of Human Tissue Kallikrein: 1—Substrate Activation in the Catalyzed Hydrolysis of H-[formula omitted]-Valyl-[formula omitted]-leucyl-[formula omitted]-arginine 4-Nitroanilide and H-[formula omitted]-Valyl-[formula omitted]-leucyl-[formula omitted]-lysine 4-Nitroanilide; 2—Substrate Inhibition in the Catalyzed Hydrolysis of Nα-p-Tosyl-[formula omitted]-arginine Methyl Ester

Hydrolysis of d-valyl-l-leucyl-l-lysine 4-nitroanilide (1), d-valyl-l-leucyl-l-arginine 4-nitroanilide (2), and Nα-p-tosyl-l-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis–Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis–Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme–substrate complex and a free substrate molecule.

아세틸콜린에스터라제 촉매에 의한 티오에스테르의 가수분해 반응 메카니즘 연구

합성된 hexanoylthiocholine을 기질로 하여 butyrylcholinesterase와의 반응을 연구하였다. 기질의 농도 변화에 따른 초기반응속도 관찰을 통해 아실기의 탄소수가 증가함에 따라 반응성은 감소하나 <TEX>$K_{m}$</TEX> 값은 0.140(mM)으로 더 강한 ES 복합체를 형성함을 알 수 있었다. Hexanoylthio-choline의 촉매화된 acetylcholinesterase 가수분해에 대한 pH-V/K profile에서 p <TEX>$K_{a}$</TEX> 값 4.974<TEX>$\pm$</TEX>0.02을 얻었다. 이는 최근 문헌의 보고와 상통하는 것으로 p <TEX>$K_{a}$</TEX> =6.2~6.4를 갖는 잔류물의 기본형에 활성이 의존하는 것으로부터 하나의 잔류물 또는 p <TEX>$K_{a}$</TEX> =4.7~5.0을 갖는 잔류물들의 촉매작용으로 계통적인 자리밀림을 보여준다. 이는 촉매화된 BChE의 활성영역 esteratic site 주변에 긴 사슬 아실기의 가수분해에 관여하는 새로운 활성영역이 존재함을 밝히는 증거이다. 분자 조형은 기질의 아실기의 탄소수에 따라 acetylcholinesterase에 의해 표현되어지는 반응과정의 변화의 합리성을 제공한다. 본 연구에서는 한국과학기술원 도핑컨트롤센터와 연계하여 acetylcholinesterase와 기질인 acylthiocholine과의 입체적으로 둘러싸인 acyl-binding site를 분자 조형하고자 노력 중에 있다. It was researched to react horse serum-BChE with hefanoylthiocholine chosen among choline esters. According as number of carbon of acyl group in choline esters was increased, reactivity was decreased but strength of ES complex was increased (Km=0,140mM). The pH-V/K profile for BChE-catalyzed hydrolysis of hexanoylthiocholine yields a p <TEX>$K_{a}$</TEX> =4.974<TEX>$\pm$</TEX>0.028. This value is equal to recent literature that shows systematic shift from dependence of activity on the basic form fo a residue that huts a p <TEX>$K_{a}$</TEX> =6.2~6.4 to catalysis by a residue or residues that has a p <TEX>$K_{a}$</TEX> =4.7~5.0. The resulting kinetic solvent isotope effect of hexanoylthiocholine is <TEX>$^{D/V}$</TEX>K=1.18. The magnitude of the isotope effect suggests that proton transfer is not an element of transition-state stabilization.n.

Interaction of 3-hydroxybenzoate with 3-hydroxybenzoate-6-hydroxylase.

The gradual quenching of the emission fluorescence of 3-HBA in the visible region upon titration with 3-HBA-6-hydroxylase and distinct changes in the near-UV circular dichroic spectrum of the enzyme in the presence of substrate suggest the formation of a stable enzyme-substrate complex. The binding of aromatic substrate 3-hydroxybenzoate to 3-hydroxybenzoate-6-hydroxylase occurs without gross changes in the backbone structure of the enzyme. The binding strength of the ES complex is partially reduced upon chemical modification of arginine, histidine, or tryptophan residues of enzyme, probably implicating their concerted action in the binding of substrate to enzyme. Partial inactivation of enzyme and diminished stability of the ES complex in response to treatment with 1 M urea could be ascribed to localized effects of the denaturant.

Correlated conformational fluctuations during enzymatic catalysis: Implications for catalytic rate enhancement.

Correlated enzymatic conformational fluctuations are shown to contribute to the rate of enhancement achieved during catalysis. Cytidine deaminase serves as a model system. Crystallographic temperature factor data for this enzyme complexed with substrate analog, transition-state analog, and product are available, thereby establishing a measure of atomic scale conformational fluctuations along the (approximate) reaction coordinate. First, a neural network-based algorithm is used to visualize the decreased conformational fluctuations at the transition state. Second, a dynamic diffusion equation along the reaction coordinate is solved and shows that the flux velocity through the associated enzymatic conformation space is greatest at the transition state. These results suggest (1) that there are both dynamic and energetic restrictions to conformational fluctuations at the transition state, (2) that enzymatic catalysis occurs on a fluctuating potential energy surface, and (3) a form for the potential energy. The Michaelis-Menten equations are modified to describe catalysis on this fluctuating potential energy profile, leading to enhanced catalytic rates when fluctuations along the reaction coordinate are appropriately correlated. This represents a dynamic tuning of the enzyme for maximally effective transformation of the ES complex into EP.

Presteady-state kinetics of Bacillus 1,3-1,4-beta-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate.

In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-beta-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s(-1), was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

Presteady-state kinetics of Bacillus 1,3–1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate

In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100s−1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme–substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

Molecular dynamics simulations of a complex of HIV-1 protease and substrate: substrate-dependent efficiency of catalytic activity

We carried out molecular dynamics (MD) simulations of the HIV-1 protease (HIV-1 PR)–substrate complex and examined the efficiency of the catalytic activity in terms of the difference in the structures of the substrates. Four kinds of substrates were used in this work: three substrate peptides composed of the sequence of the natural cleavage site by HIV-1 PR (Ac-Gln-Asn-Tyr-Pro-Ile-Val-NMe, Ac-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NMe, and Ac-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Nme) and another substrate peptide in which Leu is substituted at the P4 position (Ac-Val-Leu-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Nme). MD simulations revealed that the first three kinds of the HIV-1 PR–substrate complexes maintain a specific conformation for initiating catalytic reaction, but the last HIV-1 PR–substrate complex does not. Our results suggest that the first three substrate peptides are hydrolyzed when the respective ES complexes were formed. In contrast, a catalytic reaction seldom occurs for the last substrate peptide, even if it combined with HIV-1 PR.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor".

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.

The Role of Glutamic Acid-69 in the Activation of Citrobacter freundii Tyrosine Phenol-Lyase by Monovalent Cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.