Role of substrate binding forces in exchange-only transport systems: I. Transition-state theory

Summary An analysis of transition-state models for exchange-only transport shows that substrate binding forces, carrier conformational changes, and coupled substrate flow are interrelated. For a system to catalyze exchange but not net transport, addition of the substrate must convert the carrier from an immobile to a mobile form. The reduction in the energy barrier to movement is necessarily paid for out of the intrinsic binding energy between the substrate and the transport site, and is dependent on the formation of two different types of complex: a loose complex initially and a tight complex in the transition state in carrier movement. Hence the site should at first be incompletely organized for optimal binding but, following a conformational change, complementary to the substrate structure in the transition state. The conformational change, which may involve the whole protein, would be induced by cooperative interactions between the substrate and several groups within the site, involving a chelate effect. The tightness of coupling, i.e., the ratio of exchange to net transport, is directly proportional to the increased binding energy in the transition state, a relationship which allows the virtual substrate dissociation constant in the transition state to be calculated from experimental rate and half-saturation constants. Because the transition state is present in minute amount, strong bonding here does not enhance the substrate's affinity, and specificity may, therefore, be expressed in maximum exchange rates alone. However, where substrates largely convert the carrier to a transport intermediate whose mobility is the same with all substrates, specificity is also expressed in affinity. Hence the expression of substrate specificity provides evidence on the translocation mechanism.     

The catalytic power of enzymes: conformational selection or transition state stabilization?

The mechanism by which enzymes produce enormous rate enhancements in the reactions they catalyze remains unknown. Two viewpoints, selection of ground state conformations and stabilization of the transition state, are present in the literature in apparent opposition. To provide more insight into current discussion about enzyme efficiency, a two-state model of enzyme catalysis was developed. The model was designed to include both the pre-chemical (ground state conformations) and the chemical (transition state) components of the process for the substrate both in water and in the enzyme. Although the model is of general applicability, the chorismate to prephenate reaction catalyzed by chorismate mutase was chosen for illustrative purposes. The resulting kinetic equations show that the catalytic power of enzymes, quantified as the k(cat)/k(uncat) ratio, is the product of two terms: one including the equilibrium constants for the substrate conformational states and the other including the rate constants for the uncatalyzed and catalyzed chemical reactions. The model shows that these components are not mutually exclusive and can be simultaneously present in an enzymic system, being their relative contribution a property of the enzyme. The developed mathematical expressions reveal that the conformational and reaction components of the process perform differently for the translation of molecular efficiency (changes in energy levels) into observed enzymic efficiency (changes in k(cat)), being, in general, more productive the component involving the transition state.     

Conformational aspects of inhibitor design: enzyme-substrate interactions in the transition state.

The theory of absolute reaction rates suggests that enzymes, like other catalysts, can enhance the rate of a reaction only to the extent that they bind the altered substrate in the transition state (S++) more tightly than they bind the substrate in the ground state (S). ES dissociation constants commonly fall in the physiological range, but recent kinetic studies indicate that formal ES++ dissociation constants of less than 10(-20) M are achieved by enzymes of several classes. Studies with stable analogues suggest that these remarkable powers of discrimination involve a tendency of the enzyme to close around S++ in such a way as to maximize binding contacts; that several parts of the substrate contribute to S++ binding; and that their contributions to binding affinity can be strongly synergistic.     

Reduction of intrinsic kinetic and thermodynamic barriers for enzyme-catalysed proton transfers from carbon acid substrates

Many enzymes catalyse the heterolytic abstraction of the alpha-proton from a carbon acid substrate. Gerlt and Gassman have applied Marcus formalism to such proton transfer reactions to argue that transition states for concerted general acid-general base catalysed enolization at enzyme active sites occur late on the reaction coordinate (J. Am. Chem. Soc. 115 (1993) 11552). We postulate that as an enzyme evolves, it may decrease deltaG++ for a proton transfer step associated with substrate enolization by following the path of steepest descent on the two-dimensional surface corresponding to deltaG++, as defined by Marcus formalism. We show that for an enzyme that has decreased deltaG++ following the path of steepest descent, the values of the intrinsic kinetic (deltaG++(int,E)) and thermodynamic (deltaG(E)0) barriers for proton transfer reactions on the enzyme may be predicted from the known values of deltaG++(int,N) and deltaG(N)0 for the corresponding non-enzymic reaction and the free energy of activation on the enzyme (deltaG++(E)). In addition, the enzymic transition state will occur later on the reaction coordinate than the corresponding non-enzymic transition state (i.e. x++(E)>x++(N)) if the condition (6 - square root 2)/82deltaG++(int,N).     

Binding of reactive intermediate analogs to enzymes

A result of the fact that enzymes are catalysts is that an enzyme must bind the reactants in the transition state more tightly than the reactants in the ground state (Wolfenden, 1972; Lienhard, 1973). However, the position along the reaction co-ordinat e corresponding to the reactants in the transition state for an enzyme-catalyzed reaction does not necessarily correspond to the structure which has the highest affinity for the enzyme. Thus, analogs of structures intermediate between reactants in the ground state and in the transition state may be potent enzyme inhibitors. Bi-substrate analogs (compounds which are analogs of two substrates with the same relative orientation as the ternary enzyme complex in the ground state) are expected to have a higher affinity for many enzymes than indicated from the magnitude of the product of the binding constants of the two substrates. The value of the binding constant of a bi-substrate analog is dependent on the structure of the productive ternary complex and the kinetic nature of the reaction and is, therefore, a useful probe of the enzymic mechanism.     

New model for activation of yeast pyruvate decarboxylase by substrate consistent with the alternating sites mechanism: demonstration of the existence of two active forms of the enzyme

Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants suggested that the first transition corresponded to substrate binding in an active site and a subsequent step responsible for conversion to an asymmetric conformation. Consequently, the second enzyme state may report on "unregulated" enzyme, since the regulatory site does not participate in its generation. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substrate dependence of initial rate. The second transition corresponds to binding a substrate molecule in the regulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds to the allosterically regulated conformation, previously referred to as activated enzyme. The pH dependence of the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additional conformations of the enzyme.     

Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites

The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDeltaF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K(m). This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDeltaF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDeltaF1 to the enzyme. Specific recognition of mIIaDeltaF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.     

A substrate in pieces: allosteric activation of glycerol 3-phosphate dehydrogenase (NAD+) by phosphite dianion

The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.     

Structural basis for aconitase activity inactivation by butanedione and binding of substrates and inhibitors

Aconitase (citrate(isocitrate)hydro-lyase, EC 4.2.1.3) prior to activation demonstrates a single binding site for substrates and inhibitors. On the basis of kinetic experiments, at pH 8.5 and 37 degrees C, with monomeric butanedione in borate, this binding site was found to contain a single arginine residue. Dissociation constants at pH 8.5 and 37 degrees C, determined from inhibitory effects on butanedione inactivation rates are: citrate, 0.74 mM; D-isocitrate, 0.33 mM: cis-aconitate, 0.52 mM; tricarballytate, 0.42 mM; trans-aconitate, 0.025 mM. Corresponding dissociation constants for the active enzyme are: tricarballylate, 0.39 mM; trans-aconitate, 0.14 mM. Active site Fe2+ added to the enzyme on activation is therefore not required for binding. Km values are: citrate, 0.23 mM and cis-aconitate 0.012 mM. Binding to active enzyme is considered to be transition state binding.     

Mitochondrial GTP-AMP phosphotransferase. 2. Kinetic and equilibrium dialysis studies.

Kinetic and equilibrium dialysis substrate binding studies have been done to investigate the properties of mitochondrial GTP-AMP phosphotransferase. The results show that the enzyme has a specific requirement for divalent metal ions, namely Mg2+, Mn2+ or Ca2+ (Ca2+ is active only in the forward direction, the direction of formation of ADP). The reaction rate depends upon the ratio [Mg2+]:[substrate] rather than on the metal ion concentration alone. The enzymatic activity is influenced by NaCl (or KCl) and optimum pH occurs at 11.5 and 9.5 for guanosine and inosine nucleotides respectively. Examination of binding of substrates to the enzyme showed that there is one binding site (GTP site) for MgGTP, GTP, MgGDP or GDP per molecule of enzyme, with dissociation constants of 4.5, 4.4, 3.0, 2.2 micron respectively and one binding site (AMP site) for AMP, ADP or ATP per molecule of enzyme with dissociation constants of 20.9, 33.4 and 33.4 microns respectively. Since, within the limitations of equilibrium dialysis used in the present studies, AMP binding to one site of the enzyme could be detected only when GDP or GTP is present, the mechanism of the forward reaction may be assumed to be nearly ordered. For the reverse reaction there is no requirement of order of binding of the two nucleotides and so the mechanism of reaction may be assumed to be random.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

The mode of binding of potential transition-state analogs to acetylcholinesterase.

Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.     

Comparative affinities of the epimeric reaction-intermediate analogs 2- and 4-carboxy-D-arabinitol 1,5-bisphosphate for spinach ribulose 1,5-bisphosphate carboxylase

2-Carboxy-3-keto-D-arabinitol 1,5-bisphosphate is a tightly bound intermediate of the carboxylase reaction of ribulosebisphosphate carboxylase/oxygenase. Two stereoisomers of an analog of this intermediate, 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP) and 4-carboxy-D-arabinitol 1,5-bisphosphate (4CABP), are exceptionally potent, virtually irreversible inhibitors of the spinach carboxylase, presumably due to their structural similarity to the gem-diol (hydrated carbonyl at C-3) form of the intermediate. Incubation of the enzyme with either leads to time-dependent loss of activity. Inhibition of the enzyme is biphasic, with initial dissociation constants of 0.47 and 0.19 microM and maximal rates for tight complex formation of 2.2 and 1.8 min-1 for 2CABP and 4CABP, respectively. These values give second-order rate constants for tight complex formation of 7.8 x 10(4) and 1.6 x 10(5) M-1 s-1. To determine the overall affinity of the spinach enzyme for 2CABP and 4CABP, the release rates were determined by dual isotope exchange (3H-inhibitor complex with free 14C-inhibitor). Exchange half-times of 1.82 and 530 days were observed for 4CABP and 2CABP, respectively. Overall dissociation constants of 28 pM (2.8 x 10(-11) M) and 190 fM (1.9 x 10(-13) M) were calculated from these dissociation rates together with the rates of association determined by inactivation kinetics. The difference in affinity of 2CABP and 4CABP corresponds to 2.9 kcal/mol, presumably reflecting the difference in interaction of the enzyme with the two hydroxyls of the intermediate's gem-diol. The kinetic behavior of these two inhibitors, in particular the rather slow maximal rates of association, are consistent with the expected behavior of analogs of a labile intermediate of an enzymic reaction that is far more stable than a transition state.     

Pyruvate decarboxylase from Kluyveromyces lactis. An enzyme with an extraordinary substrate activation behaviour.

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of 1.5 mm. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied.     

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.     

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.     

Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes.

Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli. Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M). As a result, the enzyme predominantly exists in a single "gross" conformational state. Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973) Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation. The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex. Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit. In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect. CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex. Heterotropic interactions were observed for CTP but not for ATP. Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.