Isomerization couples chemistry in the ATP sulfurylase-GTPase system.

ATP sulfurylase catalyzes and couples the free energies of two reactions: GTP hydrolysis and the synthesis of activated sulfate, or APS. The GTPase active site undergoes changes during its catalytic cycle that are driven by events that occur at the APS-forming active site, which is located in a separate subunit. GTP responds to its changing environment by moving along its reaction path. The response, which may change the affinity or reactivity of GTP, can, in turn, produce alterations at the APS active site that drive APS synthesis. The resulting stepwise progression of the two reactions couples their free energies. The mechanism of ATP sulfurylase involves an enzyme isomerization that precedes and rate limits cleavage of the beta,gamma-bond of GTP. These fluorescence studies demonstrate that the isomerization is controlled by the binding of activators that drive ATP sulfurylase into forms that mimic different stages of the APS reaction. Only certain activators elicit the isomerization, suggesting that the APS reaction must proceed to a specific point in the catalytic cycle before the conformational "switch" that controls GTP hydrolysis is thrown. The isomerization is shown to require occupancy of the gamma-phosphate subsite of the GTP binding pocket. This requirement establishes that the isomerization results in a change in the interaction between the enzyme and the gamma-phosphate of GTP that emerges in the catalytic cycle during the transition from the nonisomerized to the isomerized E.GTP complex. The newly formed contact(s) appears to carry into the bond-breaking transition state, and to be essential for the enhanced affinity and reactivity of the nucleotide.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Escherichia coli alkaline phosphatase. An analysis of transient kinetics

1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.     

Kinetics of the daunomycin--DNA interaction

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.     

Proton relaxation study of the hog kidney diamine oxidase active center.

Proton relaxation studies of the interactions with hog kidney diamine oxidase of water, substrate-analogue inhibitors, and product analogues indicate that the active site Cu(II) is not located near the oxidizing site of the enzyme, rather near the nonoxidized end of the binding substrate. The studies with histamine derivatives provide evidence for a concentration-dependent occupation of two sites. The site which is populated at high concentrations provides proximity of the imadazole ring nitrogen N1 to the Cu(II). Water binds at the Cu(II) of the native enzyme. However, this water is probably not involved in the hydrolysis of the enzyme-substrate imine bond to eliminate the first reaction product. O2 does not compete with H2O for a site on the Cu(II) ion. In the case of one of the probes, namely the ammonia (product) analogue dimethylamine, the validity of the protein relaxation results was verified by also observing the nitrogen (15N) relaxation rates of ammonia itself. The conclusion that the ammonium ions is not directly bonded to the active site Cu(II) is reached from both the proton and nitrogen relaxation experiments.     

Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction). Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.     

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.     

Relaxation spectra of yeast hexokinases. Isomerization of the enzyme.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.     

Metal-dependent DNA cleavage mechanism of the I-CreI LAGLIDADG homing endonuclease

The LAGLIDADG homing endonucleases include free-standing homodimers, pseudosymmetric monomers, and related enzyme domains embedded within inteins. DNA-bound structures of homodimeric I-CreI and monomeric I-SceI indicate that three catalytic divalent metal ions are distributed across a pair of overlapping active sites, with one shared metal participating in both strand cleavage reactions. These structures differ in the precise position and binding interactions of the metals. We have studied the metal dependence for the I-CreI homodimer using site-directed mutagenesis of active site residues and assays of binding affinity and cleavage activity. We have also reassessed the binding of a nonactivating metal ion (calcium) in the wild-type enzyme-substrate complex, and determined the DNA-bound structure of two inactive enzyme mutants. The conclusion of these studies is that the catalytic mechanism of symmetric LAGLIDADG homing endonucleases, and probably many of their monomeric cousins, involves a canonical two-metal mechanism in each of two active sites, which are chemically and structurally tethered to one another by a shared metal ion. Failure to occupy the shared metal site, as observed in the presence of calcium or when the metal-binding side chain from the LAGLIDADG motif (Asp 20) is mutated to asparagine, prevents cleavage by the enzyme.     

Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis.

Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.     

Relaxation studies of enzymes: rapid isomerization in deoxyribonuclease I.

Temperature-jump relaxation studies in deoxy-ribonuclease I were carried out at 10 degrees C and [I] = 0.1 M. The single observed relaxation time, which varied from 10(-4) to 10(-5) s, was characterized as a function of enzyme concentration, pH, and indicator concentration. The concentration and pH dependences of the relaxation time are in quantitative agreement with a mechanism involving an isomerization of the enzyme coupled to a rapid proton ionization process. The best fit forward and reverse isomerization rate constants are 6.5 X 10(3) and 7.2 X 10(4) s-1, respectively; the apparent pK is 5.7. The addition of urea brought about reductions in both the amplitude of the relaxation effect and the enzyme activity.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Low-temperature studies of the sarcoplasmic reticulum calcium pump. Mechanisms of calcium binding

The mechanism of the sarcoplasmic reticulum Ca2+-ATPase was investigated at low temperatures (0 to -12 degrees C). Transient states of the enzyme were studied by two complementary techniques: intrinsic protein fluorescence and rapid filtration on Millipore filters. Intrinsic fluorescence was used to distinguish conformational states of the protein and to evaluate the rate of conversion between these states. Filtrations were used to measure the evolution of the active sites during the transition; the time resolution was 2-5 s. At sub-zero temperatures this time is shorter than the lifetime of most of the enzymatic states which have been detected. In this paper the mechanism of Ca2+ binding to the protein is investigated in the absence of nucleotides. Two basic experiments are described; (1) Kinetics of calcium binding and dissociation over a wide range of calcium concentration. (2) Kinetics of calcium exchange (45Ca2+ in equilibrium 40Ca2+) at constant concentration. The results obtained in the first series of experiments are consistent with a sequential binding to two interacting Ca2+ binding sites. Calcium ions have very fast access to a site with low apparent affinity (Kd approximately 25 microM). Occupation of this site induces a slow conformational change which increased its apparent affinity and reveals a second site of high apparent affinity. At equilibrium the two sites are not equivalent in terms of rate of exchange. Two different rates were detected k fast greater than 0.2 s-1, k slow approximately 0.015 s-1 at -10 degrees C. Removal of Ca2+ from the fast exchanging site by addition of EGTA accelerates the rate of release of the slow exchanging one. A model is proposed with two interacting Ca2+-binding sites. A set of parameters has been obtained which produces correctly the Ca2+-binding curve and the fluorescence level at equilibrium as well as the rate constants of the calcium-induced fluorescence changes over a very wide range of Ca2+ concentrations (0.02 to 150 microM). The non-equivalence of the two classes of site and the meaning of the initial low-affinity binding are discussed.     

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.     

Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin.

The binding of tri- and tetra-anionic azo dyes (Amaranth, Ponceau 4R, and Ponceau 6R) to bovine serum albumin (BSA) at pH = 7.0 and 25 degrees C has been studied by equilibrium dialysis, spectrophotometry, and by stopped-flow and temperature-jump methods. Equilibrium dialysis revealed that BSA has one primary binding site and about two secondary sites for each dye. The values of the binding constant for the primary site show that the stability of the complex at the primary site progressively increases with an increase in the number and the density of anionic charges on ligand. Kinetic data have been found to be consistent with a scheme in which a rapid bimolecular binding is followed by two isomerizations of the complex (in the case of Amaranth) or by one isomerization (in the cases of Ponceau 4R and Ponceau 6R). Equilibrium and rate constants for each step of the scheme were determined. From the results it was found that the increment of the number and the density of anionic charges on ligand accelerates the forward process of the final isomerization step but retards the backward one of it, resulting in the enhancement of the stability of the complex at the primary site. On the basis of these results and the structure of the ligands, the detailed binding mechanism has been discussed in the light of the electrostatic interaction between the ligands and the binding site on BSA.     

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Streptomyces glucose/xylose isomerase has a single active site for glucose and xylose

A kinetic method which allows one to evaluate whether an enzyme acting on two different substrates has one or two active sites was employed to study the active site of glucose isomerase which catalyses the isomerization of both glucose and xylose. The experimental data on the rates of hydrolysis of mixtures of various concentrations of glucose and xylose by the glucose isomerase from Streptomyces coincides well with the theoretical values calculated for the case of a single active site.