Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.     

End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli

Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.     

Reaction mechanism of erythrocyte phosphofructokinase.

The reaction mechanism of erythrocyte phosphofructokinase (PFK) was investigated by the initial velocity and the product inhibition. Intersecting lines obtained with initial velocity studies are consistent with a sequential mechanism and the formation of ternary complex as an intermediate. The product inhibition studies support an ordered Bi Bi mechanism in which fructose 6 phosphate (F6P) is the first substrate binding and adenosine diphosphate (ADP) is dissociated from the enzyme before fructose-1,6-P2 (FDP).     

Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica

The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.     

Human deoxycytidine kinase: kinetic mechanism and end product regulation

The kinetic properties of the monomeric deoxycytidine kinase (EC 2.7.1.74) from leukemic human T-lymphoblasts have been investigated. The results of steady-state initial-rate kinetic analysis and product inhibition studies at pH 7.5 and 37 degrees C indicate that substrate binding follows an ordered sequential pathway, with the magnesium salt of ATP being the first substrate to bind and dCMP the last product to dissociate. At subsaturating substrate concentrations, dCMP produced competitive inhibition against ATP, while against varied deoxycytidine concentrations dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either substrate. The limiting Km values for deoxycytidine and MgATP were 0.94 and 30 microM, respectively. The end product inhibitor dCTP exhibited competitive inhibition against varied ATP concentration, with a dissociation constant estimated to be 0.7 microM when extrapolated to zero ATP concentration. dCTP was purely noncompetitive against varied deoxycytidine concentration. On the basis of these kinetic results, and on the strong and specific inhibition by dCTP, it is proposed that this end product functions as a multisubstrate analogue, with its triphosphate group binding to the phosphate donor site of the enzyme and its deoxycytidine moiety overlapping and binding to the deoxynucleoside site in a highly specific manner.     

Kinetic studies of bovine liver fructose-1,6-bisphosphatase.

Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possibility. There is evidence that isomerization of the binary complex of enzyme and phosphate occurs in the kinetic mechanism. Although magnesium is required for the reverse reaction, there is no evidence to suggest that the enzyme discriminates between the magnesium-associated or divalent cation-free forms of the substrates.     

Kinetic mechanism of glucose dehydrogenase from Halobacterium salinarum

The kinetic mechanism of glucose dehydrogenase (EC 1.1.1.47) from Halobacterium salinarum was studied by initial velocity and product inhibition methods. The results suggest that both, in the forward and reverse direction, the reaction mechanism is of Bi Bi sequential ordered type involving formation of ternary complexes. NADP+ adds first and NADPH formed dissociates from the enzyme last. For the reverse direction, NADPH adds first and NADP+ leaves last. Product inhibition experiments indicate that (a), the coenzymes compete for the same site and form of the enzyme and (b), ternary abortive complexes of enzyme-NADP(+)-glucono-delta-lactone and enzyme-NADPH-glucose are formed. All the other inhibitions are noncompetitive.     

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.     

Regulation of human lymphocyte S-adenosylmethionine synthetase by product inhibition

A steady-state kinetic analysis of human S-adenosylmethionine synthetase indicates that the reaction is Bi Ter with ordered addition of ATP and L-methionine and release of S-adenosylmethionine as the first product. Pyrophosphate and phosphate are then released randomly. I-Parabolic inhibition by phosphate with respect to ATP indicates that this product must bind to more than one site. A model in which phosphate binds to the pyrophosphate site gives a rate equation that is consistent with the kinetic data. Values have been determined for those constants in the equation that are large enough to evaluate, and the in vitro kinetic behavior of S-adenosylmethionine synthetase can be predicted at substrate and product concentrations that are expected intracellularly. Inhibition by combinations of products, especially pyrophosphate and phosphate, is synergistic. Of particular interest is the ability of pyrophosphate and phosphate to increase the sensitivity of the enzyme to inhibition by S-adenosylmethionine. This phenomenon may play a role in regulating steady-state cellular concentrations of S-adenosylmethionine.     

Kinetic mechanism of AKT/PKB enzyme family

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.     

Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani

Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP(i). Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J. 18, 3533-3545). To address this issue, a steady state and transient kinetic analysis of the enzyme was performed in order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and AMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction. Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.     

Kinetic mechanisms of Ca++/calmodulin dependent protein kinases

Many of the cellular responses to Ca⁺⁺ signaling are modulated by a family of multifunctional Ca⁺⁺/calmodulin dependent protein kinases (CaMKs): CaMK I, CaMK II and CaMK IV. In order to further understand the role of CaMKs, we investigated the kinetic mechanism of CaMK II isozymes in comparison with those of CaMK I and CaMK IV by analyzing their steady state kinetics using phospholamban as a phosphoacceptor. The results indicated that (a) the CaMK family’s reaction mechanisms were of the sequential type in which all substrates must bind to enzyme before any product is released; (b) CaMK I and CaMK IV exhibited random sequential mechanism where either phospholamban or ATP can bind to the free enzyme; (c) the data of product inhibition for CaMK IIs best fit with an Ordered Bi Bi mechanism in which phospholamban is the first substrate to bind and ADP is the last product to be released; and (d) the constant α (ratio of apparent dissociation constants for binding peptide in the presence and absence of the second ligand) of all isozymes for ATP and peptide was higher than 1 indicating that the binding of phospholamban to CaMK decreased the enzyme’s affinity toward ATP.     

Kinetic studies with skeletal-muscle hexokinase

Rat skeletal-muscle hexokinase was partially purified by ammonium sulphate fractionation and gel filtration. The mechanism of the skeletal-muscle hexokinase was studied kinetically by initial-velocity analysis and product inhibition. Glucose 6-phosphate was a non-competitive inhibitor of glucose and ATP. ADP was a non-competitive inhibitor of glucose and a competitive inhibitor of ATP. The data on product inhibition and initial-velocity analysis of skeletal-muscle hexokinase support an ordered sequential mechanism (ordered Bi Bi) where the addition of substrates and release of products is in the order: ATP, glucose, glucose 6-phosphate and ADP.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Pig liver phosphomevalonate kinase: kinetic mechanism

Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate to diphosphomevalonate by ATP, one of the initial steps in the biosynthesis of steroids and isoprenoids. In previous studies, the enzyme from pig liver was purified and characterized, and preliminary work showed that the enzyme follows hyperbolic kinetics and a sequential mechanism. The present work is a more detailed analysis of its kinetic mechanism, using initial velocity and isotope exchange at equilibrium measurements. The results are compatible with a Bi Bi sequential ordered mechanism with phosphomevalonate as the first substrate and ADP the last product. The Km values estimated are 43+/-7 microM for Mg-ATP and 12+/-3 microM for phosphomevalonate, with a Vmax of 51+/-2 micromol min-1 mg of protein-1.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas

Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.     

Regulation of smooth-muscle myosin-light-chain kinase. Steady-state kinetic studies of the reaction mechanism

The kinetic mechanism of turkey gizzard smooth muscle myosin-light-chain kinase was investigated using the isolated 20-kDa light chain of myosin as substrate. The kinetic and product inhibition patterns of the forward reaction indicated an ordered sequential mechanism in which MgATP bound first, ADP was released last. The order of substrate binding and product release was confirmed independently by competitive, dead-end inhibition patterns obtained using the non-hydrolizable ATP analog adenosine 5'-[beta,gamma-imido]triphosphate. The mechanism was also characterized by a relatively strong product inhibition by ADP and a weak one by phosphorylated 20-kDa light-chain myosin, in addition to a significant inhibition by the latter product via a formation of a dead-end complex. [gamma-32P]ATP in equilibrium with [32P]phosphorylated light chain isotope-exchange data were consistent with the deduced mechanism and with the presence of the latter dead-end complex.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.