Carbamyl phosphate-dependent ATP synthesis catalyzed by formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.     

Formylation of tetrahydrofolate by formyl phosphate

Formyl phosphate is the putative intermediate in the formylation of tetrahydrofolate (THF) catalyzed by N10-formylTHF synthetase. In this study the non-enzymic reaction between formyl phosphate and THF was examined at 5 degrees C. 1H-NMR, HPLC and kinetic analysis of the proton-catalyzed conversion of the product to N5,10-methenylTHF were used to identify the product. In contrast to the enzyme reaction, which produces N10-formylTHF, N5-formylTHF was the only formylated THF derivative formed. The reaction was conducted at pH values of 3, 5, and 7, with the highest yield being obtained at pH 5 (64-85%, based on THF). The enzyme, therefore, changes the regioselectivity of this reaction by increasing the reactivity of the 10-nitrogen and either decreasing the reactivity of the 5-nitrogen or limiting its accessibility to formyl phosphate. 2-Mercaptoethanol, present in the reaction mixture to protect THF from O2, was also formylated by formyl phosphate, at the oxygen position.     

Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction.

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.     

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1)

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.     

The mechanism of dextransucrase action. Direction of dextran biosynthesis

Appropriate combinations of purified components of the reversible glycine cleavage system of rat liver catalyze three partial reactions: (1) decarboxylation of glycine or its reverse reaction catalyzed by P- and H-protein, (2) condensation of one carbon substrate and ammonia or its reverse reaction catalyzed by T- and H-protein, and (3) oxidation and reduction of active disulfide of H-protein catalyzed by L-protein. Reactions (1) and (2) give the same product which is bound to H-protein. The protein-bound product was isolated by gel filtration and converted to glycine by incubation with P-protein and CO₂ or degraded further to one carbon unit and ammonia by incubation with T-protein and tetrahydrofolate. The data are consistent with the conclusion that the enzyme-bound product is an intermediate in the reversible glycine cleavage reaction. A scheme is presented for the reactions catalyzed by the enzyme system.     

Carbamate kinase of Lactobacillus buchneri NCDO110. II. Kinetic studies and reaction mechanism

The participation of Mg2+ or Mn2+ nucleoside diphosphates in the reverse reaction catalyzed by purified carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) of Lactobacillus buchneri NCDO110 was studied. The results of initial velocity studies have indicated that Mn2+ ADP is as effective as a substrate as Mg2+ ADP is. Product inhibition studies have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate. The reaction of the enzyme with the substrates is of the random type.     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.     

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.     

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

Reversible autophosphorylation of a cyclic 3':5'-AMP-dependent protein kinase from bovine cardiac muscle.

Purified cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase of bovine cardiac muscles catalyzes the incorporation of 2 mol of 32P from [gamma-32P]ATP to seryl residues in its cAMP-binding protein. The reaction appears to be catalyzed by the protein kinase itself rather than by a protein kinase kinase and is enhanced by cAMP and by the addition of polyarginine. Phosphorylation of the purified enzyme facilitates its dissociation by cAMP (Erlichman, J., Rosenfeld, R., and Rosen, O.M. (1974) J. Biol. Chem. 249, 5000-5003) but does not affect cAMP binding. At equilibrium, 2 mol of cAMP are bound to both the phospho- and dephospho-enzymes. Phosphorylation of protein kinase is reversible. Upon addition of ADP and Mg2+, phosphate is transferred from the protein to ADP, and ATP is formed. The reverse reaction is optimal at pH 5.5 unlike the forward reaction which has a broad, more alkaline pH activity optimum. It is activated by polyarginine and dependent upon the addition of cAMP to a much greater degree than the forward reaction. The data suggest that the catalytic subunit of protein kinase catalyzes the forward and reverse reactions but do not exclude the possibility that the holoenzyme may also be active. Autophosphorylation by protein kinase and dephosphorylation by phosphrprotein phosphatases of by reverals of the autophosphorylation reaction may regulate the sensitivity of certain protein kinases to activation by cAMP in vivo.     

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.     

Site-directed mutagenesis of a potential catalytic and formyl phosphate binding site and substrate inhibition of N10-formyltetrahydrofolate synthetase

Structural studies of N(10)-formyltetrahydrofolate synthetase (FTHFS) have indicated the involvement of Arg 97 in the binding of the formyl phosphate intermediate. Two site-directed mutants were constructed to test this hypothesis: R97S (Ser substitution) and R97E (Glu substitution). The k(cat) of R97S was approximately 60% that of the wild-type enzyme and had K(m) for ATP and formate twofold higher than those of wild type. R97E was completely inactive and had a K(m) for ATP nearly six times that of wild type. Substrate inhibition by tetrahydrofolate was shown to occur in wild-type and R97S enzymes using both steady-state and transient-state kinetic approaches. These results lend greater insight into the mechanistic function of FTHFS by confirming the interaction of both ATP and formate with Arg 97 and introducing the aspect of substrate inhibition by tetrahydrofolate with regard to substrate binding and dissociation.     

Mechanism of N10-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors

N¹⁰‐formyltetrahydrofolate synthetase (FTHFS) is a folate enzyme that catalyzes the formylation of tetrahydrofolate (THF) in an ATP dependent manner. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, complexed with (1) a catalytic intermediate—formylphosphate (XPO) and product—ADP; (2) with an inhibitory substrate analog–folate; (3) with XPO and an inhibitory THF analog, ZD9331, were used to analyze the enzyme mechanism. Nucleophilic attack of the formate ion on the gamma phosphate of ATP leads to the formation of XPO and the first product ADP. A channel that leads to the putative formate binding pocket allows for the binding of ATP and formate in random order. Formate binding is due to interactions with the gamma‐phosphate moiety of ATP and additionally to two hydrogen bonds from the backbone nitrogen of Ala276 and the side chain of Arg97. Upon ADP dissociation, XPO reorients and moves to the position previously occupied by the beta‐phosphate of ATP. Conformational changes that occur due to the XPO presence apparently allow for the recruitment of the third substrate, THF, with its pterin moiety positioned between Phe384 and Trp412. This position overlaps with that of the bound nucleoside, which is consistent with a catalytic mechanism hypothesis that FTHFS works via a sequential ping‐pong mechanism. More specifically, a random bi uni uni bi ping‐pong ter ter mechanism is proposed. Additionally, the native structure originally reported at a 2.5 Å resolution was redetermined at a 2.2 Å resolution.     

Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP

Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmodulin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, kcat/KLC approximately 0.21 min-1 microM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, KmADP is approximately 30 microM and ATP is a competitive inhibitor, KIATP approximately 88 microM. For the forward reaction, measured with both isolated light chains and intact myosin, KmATP is approximately 100 microM and ADP is a competitive inhibitor, KiADP approximately 140 microM (myosin) and 120 microM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, Km = 5 microM, kcat = 1720 min-1, and reverse, Km = 130 microM, kcat = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the Km term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.     

Kinetic mechanism of ATP synthesis catalyzed by mitochondrial Fo x F1-ATPase

Initial rates of succinate-dependent ATP synthesis catalyzed by submitochondrial particles from bovine heart substoichiometrically coupled with oligomycin were found to have hyperbolic dependencies on contents of Mg x ADP, free Mg2+, and phosphate. The results suggest that Mg x ADP complex and free phosphate are true substrates of the enzyme; and an unordered ternary complex of Fo x F1-ATPase, Mg x ADP, and phosphate is generated during the catalysis. The presence of free Mg2+ is required for the reaction. Mg2+ was a noncompetitive activator of ATP synthesis relative to Mg x ADP and a competitive activator relative to phosphate. The decrease in steady-state values of Deltamu(H)+ (by the inhibition of succinate oxidase with malonate) results in the decreased value of Vmax and in a slight decrease in Km for the substrates and Mg2+ without changes in affinity for the substrates. Based on these results, a kinetic scheme of ATP synthesis is proposed.     

Cleavage of oxidized guanine nucleotide and ADP sugar by human NUDT5 protein

MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.     

A novel reaction catalyzed by unadenylylated glutamine synthetase from Escherichia coli. AMP-dependent synthesis of pyrophosphate and L-Glutamate from orthophosphate and L-glutamine

The unadenylylated, manganese form of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2 from Escherichia coli catalyzes a novel, AMP-dependent (reversible) synthesis of pyrophosphate and L-glutamate from orthophosphate and L-glutamine: Formula (See Text). The hydrolysis of the L-glutamine amide bond is coupled to the stoichiometric synthesis of pyrophosphate, although as PPi accumulates, additional hydrolysis of L-glutamine occurs in a secondary reaction catalyzed by the [manganese x enzyme x AMP x PPi] complex. The synthesis of PPi probably occurs at the subunit catalytic site in the positions normally occupied by the beta, gamma-phosphates of ATP. To promote PPi synthesis, AMP apparently binds to the subunit catalytic site rather than to the allosteric inhibitor site; equilibrium binding results suggest that Pi directs the binding of AMP to the active site. In this reaction, Mg2+ will not substitute for Mn2+, and adenylylated glutamine synthetase is inactive. Pyrophosphate is synthesized by the unadenylylated, manganese enzyme at approximately 2% of the rate of that of ATP in the reverse biosynthetic reaction. If P1 is replaced by arsenate, the enzymatic rate of the AMP-supported hydrolysis of L-glutamine is 100-fold faster than is PPi synthesis and is one-half the rate of the ADP-supported, irreversible arsenolysis of L-glutamine. This latter activity also is supported by GMP and IMP, suggesting that the catalytic site of glutamine synthetase has a rather broad specificity for the nucleotide base. The reactions supported by AMP directly relate to the mechanism of glutamine synthetase catalysis.     

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.     

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.