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.     

Kinetic mechanism of fully activated S6K1 protein kinase

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.     

Kinetics and mechanism of angiotensin phosphorylation by the transforming gene product of Rous sarcoma virus

We have studied steady state kinetics of phosphorylation of [Val5]angiotensin II by pp60src, the transforming gene product of Rous sarcoma virus. Results of initial rate studies at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 mM, respectively, and Vmax was 1.0 nmol/min/mg. The end product ADP and the ATP analog AMP-PNP were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations, but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. We also presented evidence that, while pp60src contained essential histidine and/or lysine residues in its active site, the mechanism does not involve a phosphoryl enzyme intermediate.     

Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.     

Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase

The kinetic and thermodynamic interrelationships of peptide substrate (Val5-angiotensin 11), metal-ATP, and divalent metal cations with rat liver insulin receptor tyrosine kinase (IRTK) were investigated. Results of the initial rate studies with varying peptide and MnATP substrates indicates that the kinetic mechanism for IRTK is of the sequential type and therefore rules out a ping pong Bi Bi pathway. Hence, peptide substrate and metal-ATP bind to the kinase prior to the release of products. MnADP was a linear competitive inhibitor of MnATP and a noncompetitive inhibitor of peptide substrate. A synthetic tyrosine-containing pentapeptide, Glu-Glu-Phe-Tyr-Phe (EEFYF), was a linear competitive inhibitor of peptide substrate and a noncompetitive inhibitor of MnATP. Accordingly, the data show that phosphorylation of peptide substrate occurs via a rapid random equilibrium Bi Bi mechanism in which the kinase has the potential to react initially with either of the two substrates. In contrast, divalent metal cations and metal-ATP were found to interact with the kinase in a mutually inclusive manner, with metal binding to the kinase prior to MnATP. It was also found that divalent metals increase the affinity of the kinase for metal-ATP but do not affect the affinity of IRTK for metal-ADP product. Hence, divalent metals, during the reaction of association of enzyme with one of its substrates to form the binary complex, increase the relative concentration of E-ATP complex versus E-peptide complex, thus introducing a thermodynamic-dependent ordering for the interaction of substrates with the enzyme. To investigate the thermodynamics of this system, we assumed that under initial conditions the kinetic data we obtained reflected the association constants of reactants with the enzyme.     

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.     

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.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.     

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.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.     

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.     

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k_cat for PKN1 and PKN2, and a decrease in peptide K_M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K_i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.     

Activation mechanism and steady state kinetics of Bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.     

Kinetic studies of the reaction mechanism of rat liver phosphoglycerate kinase in the direction of ADP utilization

The kinetic properties of rat liver phosphoglycerate kinase were investigated in the forward direction of the reaction (utilization of ADP). The kinetic studies were performed in an assay system using combined hexokinase/glucose-6-phosphate dehydrogenase as an ATP trap. The Km values for Mg ADP1- and 1,3-diphospho-D-glycerate were approximately 0.11 and 0.006 mM, respectively. Reciprocal plots of 1/v versus 1/ (Mg ADP1-) at different fixed concentrations of 1,3-diphospho-D-glycerate and 1/v versus 1/ (1,3-diphospho-D-glycerate) at different fixed concentrations of Mg ADP1- were apparently parallel. However, product inhibition studies (3-phospho-D-glycerate), dead-end inhibition studies (2,3-diphospho-D-glycerate), and adenosine and AMP inhibition patterns yielded results consistent with a rapid equilibrium random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the second substrate. Existence of two sites for 3-phospho-D-glycerate is suggested.     

Purification and properties of multiple isoforms of a novel thiol methyltransferase involved in the production of volatile sulfur compounds from Brassica oleracea

Five functional isoforms of a novel plant thiol methyltransferase from the leaves of cabbage (Brassica oleracea L.) were purified to electrophoretic homogeneity. Pooled, partly purified preparations of the enzyme were previously shown to methylate thiol compounds released upon the hydrolysis of glucosinolates. The enzyme could also accept halide ions as substrates. The estimated molecular masses of the purified isoforms ranged between 26 and 31 kDa. The three most abundant isoforms of the enzyme could all catalyze the S-adenosyl-l-methionine-dependent methylation of thiocyanate, a number of organic thiols and iodide. However, the kinetic properties of these forms toward various substrates differed widely. None of the isoforms examined methylated the O- and N-equivalents of the thiol substrates. The three isoforms also had distinct pH optima, covering the range from 5 to 9. Their kinetic analysis indicated that they shared a sequential substrate binding mechanism and an Ordered Bi Bi mechanism for substrate binding and product release. Partial internal amino acid sequence from one isoform showed high similarity to an Arabidopsis EST of unknown function, and to a recently cloned methyl chloride transferase from Batis maritima. The differences in the pH optima and kinetic properties of the isoforms suggest that each may methylate a specific substrate or a narrow group of substrates under cellular conditions.     

Purification and characterization of homo- and hetero-dimeric acetate kinases from the sulfate-reducing bacterium Desulfovibrio vulgaris

Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (alpha(S)(2)) and the other (AK-II) was a heterodimer (alpha(S)alpha(L)). On SDS-PAGE, alpha(L) and alpha(S) subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg(2+) requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.     

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.