Enzyme relaxation in the reaction catalyzed by triosephosphate isomerase: detection and kinetic characterization of two unliganded forms of the enzyme

Triosephosphate isomerase has been shown to exist in two unliganded forms, one of which binds and isomerizes (R)-glyceraldehyde 3-phosphate and the other of which binds and isomerizes dihydroxyacetone 3-phosphate. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations and yields a rate constant of about 10(6) s-1 for the interconversion. Although the molecular nature of the two forms of unliganded enzyme is not defined by these experiments, a shuffling of protons among active site residues, or a protein conformational change, or both, may be involved. This study, coupled with the known rate constants for the substrate-handling steps of triosephosphate isomerase catalysis, completes the kinetic characterization of the catalytic cycle for this enzyme.     

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.     

Reaction mechanism and structure of the active site of proline racemase.

Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.     

Energetics and mechanism of proline racemase

The results from the previous six papers are collated so as to allow the construction of the complete free energy profile for the reaction catalyzed by proline racemase. This profile includes the step that involves the isomerization of the two forms of free enzyme, which can become rate limiting at very high substrate levels (in "oversaturation"). The mechanism of the reaction has been defined, the results being best accommodated by a route that involves a transition state or unstable intermediate in which the proline carbanion is flanked by the two catalytic thiols of the enzyme.     

The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.

The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.     

Energetics of enzyme catalysis. I. Isotopic experiments, enzyme interconversion, and oversaturation

An enzyme-catalyzed interconversion of one substrate, S, and one product P, by an enzyme that exists in two forms E1 and E2 where E1 binds S and E2 binds P, is considered S + E1 in equilibrium E1S in equilibrium E2P in equilibrium E2 + P. Under reversible conditions (where the concentrations of S and P are not far removed from their equilibrium values) it is shown that, in addition to the usual unsaturated and saturated behaviour there exists a third regime at high substrate concentration: the oversaturated region. In this region, the rate-limiting transition state is the interconversion of the unliganded forms of the enzyme: E1 and E2. Expressions for six different experiments involving deuterium, tritium and 14C labels are presented. By considering the results from these experiments, the nature and importance of the enzyme interconversion steps can be elucidated.     

Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis

An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).     

Energetics of enzyme catalysis. II. Oversaturation, case diagrams, reversible and irreversible behaviour

Most enzymes react in vivo under reversible conditions where the substrate and product concentrations are not far removed from equilibrium values. Under these conditions when the concentration of substrate is increased, in addition to the usual unsaturated and saturated behaviour we find a third type of kinetic regime at high substrate concentration-oversaturation. In this regime the rate limiting transition state involves interconversion of free enzyme forms. For a one substrate/one product enzyme, case diagrams can be constructed which depict the kinetic behaviour as a function of substrate and product concentrations. Six different cases are found and are discussed with the relevant free energy profiles. A systematic procedure is described for the investigation and construction of the case diagram.     

Enzymes as biosensors. 1. Enzyme memory and sensing chemical signals

When a free enzyme exists under different conformations that 'slowly' isomerize during the conversion of a substrate into a product, the corresponding 'slow' relaxation component may interfere with the steady-state component. The apparent steady-state rate that may be measured under these conditions is called the meta-steady-state rate for it refers to the existence of metastable states of the enzyme during the reaction. By contrast to the real steady-state rate, the meta-steady-state rate is dependent upon the initial state of the enzyme, that is on the respective concentrations of the free enzyme forms. The simplest model that may display this type of behaviour is the mnemonical model. For a fixed concentration of the last product of the reaction sequence the meta-steady state is different depending on that concentration being reached by an increase or a decrease of a previous concentration. This means that the meta-steady-state rate describes a hysteresis loop as the product concentration is increased and decreased. Owing to the existence of metastable states, the enzyme system behaves as a biosensor that is able to detect both a concentration and the direction of a concentration change. The existence of the hysteresis loop of the meta-steady-state rate implies that the two free enzyme forms display hysteresis as well. A chemical potential, called the sensing potential, is specifically associated with the 'perception' of the direction of the thermodynamic force generated by the decrease or the increase of the concentration of the ligand that binds to one of the enzyme conformations. The sensing potential of the enzyme conformer that does not bind the product increases and reaches a plateau as the chemical potential of that product is raised. Alternatively the sensing potential of the other conformer vanishes at low and high chemical potentials of the product and is significant for intermediate chemical potentials. Enzymes that display very slow conformation changes may thus be viewed as elementary sensor devices.     

Investigating the geminal diamine intermediate of Yersinia pestis arginine decarboxylase with substrate, product, and inhibitors using single wavelength stopped-flow spectroscopy

The reaction mechanism of Yersinia pestis arginine decarboxylase has been investigated using a series of substrate, product, and inhibitors. Using single wavelength stopped-flow spectroscopy, novel mechanistic features were noted in the presence of the product, agmatine. By focusing on the excitation and emission wavelengths of the geminal diamine intermediate, we were able to monitor the formation and decay of two different geminal diamine species. Experiments revealed that the enzyme exists in two different conformational states--one that binds ligand and one that does not. The on and off rates for the conversion between the two conformational states was determined to be 390 s-1 and 880 s-1, respectively. The KD for agmatine binding was 6 mM. In addition, experiments revealed a pH-dependent conversion between two states of the enzyme. The deprotonated form of the enzyme binds ligand more slowly than the protonated form. The rates for the formation of the geminal diamine and external aldimine in this pathway were determined to be 25 and 4 s-1, respectively. There is also a slow interconversion between the protonated and deprotonated enzymes that has a pKa of approximately 8.0. Finally, the formation of the geminal diamine was determined to be Mg2+-dependent.     

Inhibition of homogeneous human eosinophil arylsulfatase B by sulfidopeptide leukotrienes

Arylsulfatase B, purified to homogeneity from human eosinophils, is a tetrameric enzyme whose activity varied in accordance with the state of association of its monomeric subunits. The rate of dissociation of oligomeric forms was slow relative to the rate of the enzymatic reaction so that the kinetic properties of the enzyme depended on the concentration of the enzyme before assay. For concentrated enzyme solutions (14 micrograms/ml), Lineweaver-Burk analysis demonstrated substrate inhibition at greater than or equal to 20 mM substrate and revealed two distinct regions of activity at low and intermediate substrate concentrations. The addition of bovine serum albumin (60 micrograms/ml) or sucrose (0.25 M), which prevent subunit dissociation, yielded a linear relationship on Lineweaver-Burk analysis at non-inhibitory substrate concentrations. For dilute enzyme concentrations (4.7 micrograms/ml), inhibition occurred at greater than or equal to 2 mM substrate. Nanomolar amounts of leukotriene C4 (LTC4), relative to millimolar concentrations of substrate, inhibited eosinophil arylsulfatase B. On Lineweaver-Burk analysis, the pattern of inhibition of LTC4 with concentrated enzyme was compatible with competitive inhibition of only one oligomeric form of the enzyme, whereas at low enzyme concentrations the pattern of inhibition was apparently competitive. These findings suggest that LTC4 is a potent competitive inhibitor of a dissociated, possibly dimeric, form of the enzyme. Nanomolar concentrations of LTC4, leukotriene D4, and leukotriene E4 were equally inhibitory, whereas leukotriene B4 and isomeric 5,12-dihydroxyeicosatetraenoic acids had no inhibitory activity, indicating a requirement for a thiopeptide at C-6. Thiopeptide leukotriene analogs without an intact triene structure also lacked inhibitory activity. Sulfoxide analogs of LTC4 and leukotriene D4 were potent inhibitors, although two sulfone analogs of leukotriene D4 were not inhibitory. Arylsulfatase B did not inactivate the spasmogenic activity of sulfidopeptide leukotrienes. These findings indicate that sulfidopeptide leukotrienes and their sulfoxide derivatives may regulate by competitive inhibition the activity of oligomeric forms of the eosinophil lysosomal hydrolase, arylsulfatase B.     

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.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase of Rhodospirillum rubrum: effects of Mg2+, phosphate, and pyrophosphate

The relation that exists between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for added Mg2+. Optimal rates of hydrolysis were attained at 1 mM Mg2+ with 0.67 mM pyrophosphate; the rate od hydrolysis correlated with the concentration of Mg-pyrophosphate, which indicated that the latter was the substrate for hydrolysis. The Pi-PPi exchange reaction rate was low at concentrations of added Mg2+ below 1 mM (0.67 mM pyrophosphate), but increased as the concentration of Mg2+ in the medium was increased. The Pi-PPi exchange reaction depends on the concentration of MgHPO4, which suggests that this is the substrate in the exchange reaction. However, it is likely that free Mg2+ also exerts a favorable effect on the Pi-PPi exchange reaction. The optimal concentration for the Pi-PPI exchange reaction was approx 240 microM, which suggests that the concentration of the hydrolyzable substrates modulates the kinetic characteristics of the enzyme.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Kinetic model of the functioning of pyruvate kinase from bovine adrenal cortex

The dependence of pyruvate kinase reaction rate on the concentration of one of the ligands--ADP or MgCl2--at constant concentrations of the other ligand was studied. The enzyme activity vs ligand concentration curves have fairly symmetrical peaks which correspond to the range of approximately equal ligand concentrations. The S-shaped dependence is observed only over the range of concentrations close to the dissociation constant for the Mg-ADP- complex (0.7 mM) under the given experimental conditions. The data obtained are consistent with the results of the first model kinetics within the framework of the London-Steck theory. The substrate for pyruvate kinase is the Mg-ADP- complex, while free Mg2+ and ADP3- competitively inhibit the enzyme. The inhibition constants are equal to 44 and 1 mM, respectively. The inhibiting effects of the metal and dinucleotide may be due to the competition with the substrate for the enzyme active site. Taking into consideration the fact that the binding of one of the ligands to the enzyme depends on the presence of the other ligand, a conclusion is drawn that Mg2+ forms a bridge with ADP3- and pyruvate kinase from adrenal cortex.     

Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models

To delineate better the reaction sequence of the (Na+ + K+)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na+ + K+)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after Pi release. However, contrary to requirements of usual formulations, Pi is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K+, and with exclusive binding of substrate vs. either Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-K+ enzyme form occurring in the (Na+ + K+)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E1/E2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E2 conformation as the active site for the phosphatase reaction.     

Subunit interactions in enzyme transition states--antagonism between substrate binding and reaction rate

The principles of structural kinetics as applied to polymeric enzymes have been reinvestigated in order to take account of the probable existence of subunit interactions in the enzyme transition states. On the basis of simple and plausible postulates, structural rate equations have been derived for dimeric enzymes and compared to substrate binding isotherms. It then becomes possible to understand how subunit interactions affect substrate affinity and enzyme reaction rate. There exists an antagonism between substrate binding to the enzyme and the steady state rate of product appearance. If subunit interactions increase the rate of product appearance, they decrease the fractional saturation of the enzyme by the substrate. Alternatively, if they decrease the reaction velocity they increase the fractional saturation. This seemingly paradoxical effect is the direct consequence of subunit interactions occurring in both the ground and the transition states.     

A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase.

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.