An amine: hydroxyacetone aminotransferase from <Emphasis Type="Italic">Moraxella lacunata</Emphasis> WZ34 for alaninol synthesis

An amine:hydroxyacetone aminotransferase from an isolated soil bacterium, Moraxella lacunata WZ34, was employed to synthesize alaninol in the presence of hydroxyacetone and isopropylamine in this study. The optimal carbon and nitrogen sources were glycerol and beef extract, respectively. A wide range of amino donor specificity was detected with the aminotransferase, which exhibited a relative high activity (9.83 U mL(-1)) in the presence of isopropylamine. The enzyme was the most active at pH 8.5, and showed relatively higher activity at alkaline than acidic pH. Maximum activity was achieved at 30 degrees C, and the enzyme had good thermal stability below 60 degrees C. Metal ions such as Mg(2+) had positive effect (132.6%) on the enzyme, and (aminooxy)acetic acid, a typical aminotransferase inhibitor, significantly inhibited its activity. The enzyme activity was enhanced by the addition of 0.05 mM pyridoxal-5'-phosphate (PLP).     

Conformational changes of active site of copper zinc superoxide dismutase can be detected sensitively by electron-transfer reaction

The electron-transfer (ET) reaction between Fe(CN)64- and copper zinc superoxide dismutase (CuZn-SOD) occurs at the active site of the enzyme. The ET parameters which are sensitive to the denaturation have been used to determine the conformational changes of the active site induced by guanidine hydrochloride and thermal denaturation. The decreases of ET rates for all the denatured enzyme samples reflect the collapse of the active cavity of enzyme in the unfolding processes. The interesting changes of ET amplitude for the enzyme denatured at different pH values suggest that electrostatic interaction plays an important role in the conformational changes of active site. From the results of the kinetic analyses, it is concluded that the conformational changes of the active site are parallel with the inactivation.     

Bile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.     

变性过程中铜锌超氧化物歧化酶的活性通道

在酶的盐酸胍变性和热变性过程中,尝试采用电荷传递反应分析方法和电子自旋共振方法考察了酶活性部位的构象变化。酶活力与构象的变化行为表明,酶的活性部位通道先于酶分子的整体构象而发生变化,它是与酶的失活同时发生的。尽管酶活性部位中的金属离子保证了酶较高的稳定性,但酶的活性部位,特别是活性通道仍然是相对脆弱的。     

Enzyme adaptation to alkaline pH: atomic resolution (1.08 A) structure of phosphoserine aminotransferase from Bacillus alcalophilus

The crystal structure of the vitamin B(6)-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution. The model was refined to an R-factor of 11.7% (R(free) = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5'-phosphate are replaced, and an additional hydrogen bond to the O3' atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional beta-strands in a surface loop with a third beta-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.     

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.     

pH-Dependent conformational stability of the ribotoxin alpha-sarcin and four active site charge substitution variants

Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.     

Muscle contraction: viscous-like frictional forces and the impulsive model

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.     

Guanidine-induced equilibrium unfolding of a homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT)

4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity.     

Effects of activators on chemically modified yeast hexokinase.

Enzymic studies performed with chemically modified yeast hexokinase (ATP : D-hexose-6-phosphotransferase) confirm previous results indicating that the sulfhydryl, imidazol and most of the reactive amino groups do not seem to be directly implicated in the enzyme active site. On the other hand the modification of these functional groups of the enzyme does not affect the transition between the acidic inactive form to an active enzyme form after deprotonation. The chemically modified forms of hexokinase and the native enzyme are affected in the same way by activators (citrate, D-malate, 3-phosphoglycerate and Pi) when the activity was measured at pH 6.6. Moreover the loss of enzyme activity observed in the course of the chemical modifications is accompanied by an increase of the activation effect. This increase must be related to some reorganization of the enzyme active site in presence of the effectors, since the same effect was observed when hexokinase was denatured with 3M urea at pH 7.5. However no increase in the activation effect was observed when the denaturation was carried out at pH 6.5 At this pH the loss in activity and the change of optical absorption at 286 nm were much slower than at pH 7.5, which indicates a great difference in the protein structure between these pHs.     

Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage

The X-ray susceptibility of the lysine-pyridoxal-5'-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 A resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5'-phosphate and the Lys residue. Analysis of the "undamaged" structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 x 10(6) Ggamma. Our data provide new insights into the enzymatic activation of pyridoxal-5'-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.     

4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate

Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.     

Adenylation-dependent conformation and unfolding pathways of the NAD+-dependent DNA ligase from the thermophile Thermus scotoductus

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.     

Conformational stability of pig citrate synthase and some active-site mutants

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.     

Temperature, organic solvent and pH stabilization of the neutral protease from Salinovibrio proteolyticus: significance of the structural calcium

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.     

Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.     

The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability

Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein folding.     

Conformation and stability of elastase from Atlantic cod, Gadus morhua

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.     

Kinetic and structural characterization of reversibly inactivated beta-lactamase

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.