Role of Magnesium in the Binding of Substrate and Effectors to Phosphoenolpyruvate Carboxylase from a CAM Plant

The binding of phosphoenolpyruvate, malate, and glucose 6-phosphate to phosphoenolpyruvate carboxylase purified from Crassula argentea Thunb. was measured using both the intrinsic tryptophan fluorescence of the enzyme and the extrinsic fluorescence of the complex of 8-anilino-1-napthalenesulfonate with the enzyme. It was found that the substrate phosphoenolpyruvate can bind in the absence of magnesium but is bound in greater quantities and more tightly when magnesium is present. Malate reduces the binding of phosphoenolpyruvate, while glucose 6-phosphate increases the binding of the substrate. Glucose 6-phosphate requires magnesium to bind to the enzyme, while malate does not. The general trends from the binding experiments using fluorescence methods were confirmed by activity determinations using assays performed in the absence of magnesium.     

Purification and properties of NADP-specific malate dehydrogenase from bovine adrenal cortex mitochondria

An electrophoretically homogeneous preparation of mitochondrial NADP-dependent malate dehydrogenase with a specific activity of 155 u./mg and a 67% yield has been obtained, using ammonium sulfate fractionation, gel filtration through Toyopearl HW-55 F, ion-exchange chromatography on DEAE-Toyopearl 650 M and affinity chromatography on 2',5'-ADP-Sepharose 4B. The molecular mass of native malate dehydrogenase is 260 kD; Mr of the SDS-treated enzyme is 61 kD, which is suggestive of a tetrameric structure of the protein. Malate dehydrogenase is active only in the presence of Mg2+ or Mn2+, but not Ca2+ or Ba2+. The Km' values for Mn2+ and Mg2+ are 50 and 66 microM, respectively. At low malate concentrations and NADP saturation, the enzyme is characterized by a sigmoidal kinetics which changes to hyperbolic at low concentrations of NADP. The Lineweaver--Burk plots for the dependence of the initial reaction rate on the concentration of one substrate at several fixed concentrations of the other substrate intersect to the left of the B-axis. NADPH competes with NADP:pyruvate inhibits malate dehydrogenase ++noncompetitively with respect to the coenzyme. NADPH and pyruvate inhibit the malate dehydrogenase-catalyzed reaction via a mixed type mechanism with respect to malate. The data obtained are consistent with a consecutive mechanism of reaction, whose first substrate is NADP and the last product is NADPH.     

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.     

Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.     

Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.     

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.     

Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum

Malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum is purified 50-fold to electrophoretic homogeneity. The purified enzyme crystallizes readily. Native malate dehydrogenase shows a relative molecular mass of 144 000. It is a tetramer of identical subunits with a relative molecular mass of 36 600. Malate dehydrogenase from Thermoplasma uses both NADH and NADPH as coenzyme to reduce oxaloacetate. The enzyme shows A-side (pro-R) stereospecificity for both coenzymes. The pH optimum for the reduction of oxaloacetate in the presence of NADH is found to be at pH 8.1. At pH 7.4 the Km value for oxaloacetate is found to be 5.6 microM while for NADH a value of 11.7 microM is found. The homogeneous enzyme shows a turnover number of kcat = 108 s-1.     

Properties and function of mitochondrial malate enzyme from bass liver

Malate enzyme (L-malate: NADP+ oxidoreductase oxaloacetate decarboxylating, EC 1.1.1.40) from bass liver mitochondria was purified to over 90% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight estimated by gel filtration was 316,000. Analysis of the enzyme on sodium dodecylsulphate-polyacrylamide disc gel electrophoresis was shown to be a tetramere protein. The enzyme required bivalent cations for catalysis, (Mn2+ or Mg2+) and displayed a narrow pH optimum (8.4-8.6 for Tris-HCl buffer) and was inactivated by p-chloromercuribenzoate. The double reciprocal initial velocity plots of both of the substrates, NADP and malate, were linear and intercepting at a point that suggests a sequential mechanism. Product inhibition studies with NADP and malate as variable substrate are consistent with an ordered Bi-Ter mechanism.     

Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.     

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus

Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Spectroscopic evidence for ligand-induced conformational change in NADP+:isocitrate dehydrogenase

Conformational changes induced by binding of ligands to cytosolic NADP(+)-specific isocitrate dehydrogenase from lactating bovine mammary gland were assessed using circular dichroism and fluorescence techniques. The secondary structure of isocitrate dehydrogenase, as monitored by CD spectra in the far-UV region, is unaltered by enzyme-ligand interactions; in contrast, dramatic changes occur in the near-UV region (270-290 nm) assigned to tyrosine and/or solvent-exposed tryptophan residues. Both the coenzyme analog, 2'-phosphoadenosine 5'-diphosphoribose, and NADPH have an effect on the CD spectrum which is opposite to that produced by metal complexes of either isocitrate or citrate. A CD band at 292 nm assigned to approximately 2 tryptophan residues in a hydrophobic environment is unchanged by binding of substrate or coenzyme. Approximately 30% of the intrinsic fluorescence of isocitrate dehydrogenase, corresponding to approximately 2 tryptophan residues, is not quenched by acrylamide in the absence of 6.3 M guanidine hydrochloride and remains unquenched in the enzyme-substrate complex. The constancy in the proportion of buried and exposed tryptophan residues implicates tyrosine in the observed near-UV CD spectral changes. Since binding of ligands does not influence quaternary structure (Seery, V.L., and Farrell, H. M., Jr. (1989) Arch. Biochem. Biophys. 274, 453-462), activation of isocitrate dehydrogenase may be related to a substrate-induced conformational transition.     

Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine

Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.     

Lactate dehydrogenase-induced conformational changes of F-actin in myosin-free ghost single fibres

The changes in conformation of F-actin induced by the binding of the glycolytic enzyme lactate dehydrogenase were studied in myosin-free single ghost muscle fibres. The formation of the lactate dehydrogenase-F-actin complex was accompanied by changes in the parameters of intrinsic (tryptophan) and extrinsic (rhodaminyl-phalloin) polarized fluorescence of ghost muscle fibre F-actin. Lactate dehydrogenase stimulated actin-activated Mg2+-ATPase of myosin subfragment 1 by 30%. F-actin of ghost fibres depressed lactate dehydrogenase activity to 20% of the initial values. It is suggested that the energy-providing mechanism is coupled with that of muscle contraction through conformational changes in F-actin.     

Effect of thiol reagents on the activity of NADP-dependent malate dehydrogenase isolated from bovine adrenal cortex cytoplasm

NADP-dependent malate dehydrogenase was rapidly inactivated in the presence of mercurous chloride. Titration of malate dehydrogenase by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in a solution of 8 M urea revealed 18 SH groups per molecule of the enzyme. Eight sulphydryl groups reacted with DTNB in native malate dehydrogenase and their modification was not accompanied by a loss of the enzyme activity. The interaction of p-chloromercury benzoate (PCMB) with malate dehydrogenase resulted in a 70% decrease in the enzyme activity. The binding of the thiol reagents by the malate dehydrogenase molecule appreciably increased the Michaelis constant value for the substrate. In the presence of magnesium ions, NADP and malate did not affect the process of malate dehydrogenase modification by DTNB and did not protect the enzyme from the inactivation by PCMB. It is suggested from the data obtained that the sulphyryl groups are involved in maintaining the active conformation of the enzyme.     

Induced fit and kinetic mechanism of adenylation catalyzed by Escherichia coli threonyl-tRNA synthetase

Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.