A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.     

Slow tight binding inhibition of proteinase K by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.     

Subunit interactions in the methionyl-tRNA synthetase of Bacillus stearothermophilus.

The methionyl-tRNA synthetase from Bacillus stearothermophilus is shown to be a dimer of 2 x 82,000 with identical subunits. It exhibits negative cooperativity in substrate binding and "virtual" halt-of-the-sites reactivity. The enzyme binds only 1 mol of methionine in the absence of other ligands, but several methods show that 2 mol of methionyl adenylate are bound per enzyme dimer. However, one of these adenylates is formed 480 times faster than the other (k1 = 29 sec-1 and k2 = 0.06 sec-1). While the rapid phase of the reaction follows normal saturation kinetics with respect to substrate concentration, the rate of the slow phase is independent of substrate concentrations down to 1 muM. It is suggested that the very slow rate of formation of the second adenylate reflects a rate limiting conformational change which precedes a more rapid chemical step on the second subunit.     

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.     

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.     

Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occurring mutations

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in k(cat)/K(0.5)(h)) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K(i)) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.     

Glucose modulation of glucokinase activation by small molecules

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K 0.5 ( approximately 1 mM vs approximately 8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the k cat to various degrees (62-95% of the control activity). These activators relieved GK's inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose K D and the steady-state parameter K 0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric binding site which is present in the glucose-induced, active enzyme form of GK and absent in the inactive apoenzyme [Kamata et al. (2004) Structure 12, 429-438]. A mechanistic model that brings together the kinetic and structural data is proposed which allows qualitative and quantitative analysis of the glucose-dependent GK regulation by small molecules. The regulation of GK activation by glucose may have an important implication for the discovery and design of GK activators as potential antidiabetic agents.     

Crystal structure of E339K mutated human glucokinase reveals changes in the ATP binding site

Human glucokinase (GK) plays an important role in glucose homeostasis. An E339K mutation in GK was recently found to be associated with hyperglycemia. It showed lower enzyme activity and impaired protein stability compared to the wild-type enzyme. Here, we present the crystal structure of E339K GK in complex with glucose. This mutation results in a conformational change of His416, spatially interfering with adenosine-triphosphate (ATP) binding. Furthermore, Ser411 at the ATP binding site is phosphorylated and then hydrogen bonded with Thr82, physically blocking the ATP binding. These findings provide structural basis for the reduced activity of this mutant.     

Kinetic and thermodynamic studies of tet repressor-tetracycline interaction

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg(2+) ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix-turn-helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg(2+) can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 x 10(5) M(-)(1) s(-)(1) and 2.1 s(-)(1) for the forward and backward reaction, respectively, and gave the binding constant K(a) = 0.96 x 10(5) M(-)(1). The kinetics of [Tc-Mg](+) binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k(a) = 1.4 x 10(5) M(-)(1) s(-)(1) and k(d) = 2.2 x 10(-)(2) s(-)(1) and binding constant K(a) = 6.3 x 10(6) M(-)(1). The first step of [Tc-Mg](+) association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k(c), and backward, k(-)(c), reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg(2+) to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 degrees C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K(a) = 9.0 x 10(6) M(-)(1) and K(a) = 7.0 x 10(4) M(-)(1) for Tc with and without Mg(2+), respectively. The binding of the inducer to TetR, in the presence and absence of Mg(2+) ion, is an enthalpy-driven reaction characterized by DeltaH = -51 kJ mol(-)(1) and DeltaH = -33 kJ mol(-)(1), respectively. The entropy change, DeltaS, for the interaction in the presence of Mg(2+) is equal to -38.9 J K(-)(1) mol(-)(1), and for the tetracycline alone, it was estimated at -17.6 J K(-)(1) mol(-)(1).     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Thermal stabilty of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea

We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.     

Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: temperature sensitivity and effects of ADP.

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme.

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1). At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1). From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex. In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).     

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.     

Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions

The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.     

Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

Abstract

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with K_i and α values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with K_i and α values of 0.4μM and 0.15, respectively. LY83583 and 2-anilino-l,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar K_i values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a K_i value of 11 μM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by pre-incubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.     

Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 10 s(-1)). The release rate (or protonation of the enzyme GSH thiolate complex) of GS(-) was slow (k(-2) = 0.016 s(-1)), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E*GS(-) complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378-3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly coupled, because proton release (as well as k(cat)) was found to be approximately 4 times slower than GSH thiolate formation in an unbuffered system. Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k(2)) for thiolate formation and k(cat) (in the reaction with 1-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1. This indicates that thiolate formation contributes to k(cat) for the activated enzyme, as suggested previously for unactivated MGST1. The stoichiometry of thiolate formation, proton release, and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.     

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP.

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C.