Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate,Pi dikinase

Pyruvate,Pi dikinase regulatory protein (PDRP) has been highly purified from maize leaves, and its role in catalyzing both ADP-mediated inactivation (due to phosphorylation of a threonine residue) and Pi-mediated activation (due to dephosphorylation by phosphorolysis) of pyruvate,Pi dikinase has been confirmed. These reactions account for the dark/light-mediated regulation of pyruvate,Pi dikinase observed in the leaves of C4 plants. During purification to apparent homogeneity the ratio of these two activities remained constant. The molecular weight of the native PDRP was about 180,000 at pH 8.3 and 90,000 at pH 7.5. Its monomeric molecular weight was 45,000. It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation. Michaelis constants for orthophosphate and the above form of active pyruvate,Pi dikinase were determined, as well as the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi. For the inactivation reaction, Km values were 1.2 microM for the active pyruvate,Pi dikinase and 52 microM for ADP. CDP and GDP but not UDP could substitute for ADP. The inactivation reaction is inhibited by inactive pyruvate,Pi dikinase competitively with respect to both active pyruvate,Pi dikinase and ADP. Both the activation and inactivation reactions catalyzed by PDRP have a broad pH optimum between 7.8 and 8.3. The results are discussed in terms of the likely mechanism of dark/light regulation of pyruvate,Pi dikinase in vivo.     

Pyruvate,orthophosphate dikinase from Acetobacter aceti

A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg²⁺ was required (forward K _m 1.70 mM; reverse K _m 0.87 mM) and no other divalent cation was able to replace it. The K _m values for pyruvate, ATP, and P_i were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K _m values for PEP, AMP, and PP_i were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, P_i-PP_i were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione     

Properties and mechanism of action of pyruvate, phosphate dikinase from leaves

1. Sugar-cane leaf pyruvate,P(i) dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and P(i) into phosphoenolpyruvate, AMP and PP(i). 2. The purified enzyme was stable at pH8.3 only if stored at about 20 degrees in the presence of Mg(2+) and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PP(i) were 0.11mm and 0.04mm respectively and that for AMP was less than 4mum. 4. At pH8.3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PP(i) were derived from P(i) and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps:Enzyme+ATP+P(i) right harpoon over left harpoon Enzyme-P+AMP+PP(i)Enzyme-P+pyruvate right harpoon over left harpoon Enzyme+phosphoenolpyruvate     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii.

Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.     

Steady-state investigations of the mechanism of histidinol dehydrogenase.

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.     

Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase.

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.     

Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays)

Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C₄ plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C₄ photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP⁺-malate dehydrogenase proceeded more rapidly than photosynthesis.     

Cloning of cDNA for pyruvate, Pi dikinase from maize leaves.

To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E. coli, strain RR1. Colonies were screened by sequential hybridization with 32P-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)+RNA fractionated by sucrose density centrifugation. Among the clones bearing cDNA homologous to high molecular weight poly(A)+RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf. Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits. Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.     

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30°C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C₄ plants.

Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Chemical modification of the bifunctional regulatory protein of maize leaf pyruvate,orthophosphate dikinase. Evidence for two distinct active sites

The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.     

Regulation of C4 photosynthesis catalytic dephosphorylation and Pi-mediated activation of pyruvate Pi dikinase

In experiments designed to test the reversibility of ADP-dependent inactivation and Pi-dependent activation of pyruvate, Pi dikinase , it was found that the preferred substrate for Pi dependent activation is the catalytically non-phosphorylated form of pyruvate, Pi dikinase . Only the second of the two partial reactions catalysed by pyruvate, Pi dikinase is inhibited when pyruvate, Pi dikinase is inactivated by ADP-dependent phosphorylation. Neither ADP-dependent inactivation nor Pi-dependent activation reactions were found to be reversible.     

Partial characterization of the in vitro activation of inactive pyruvate, pi dikinase from darkened maize leaves

In vitro activation of dark-inactivated pyruvate, orthophosphate dikinase extracted from maize (Zea mays L. cv. Golden Cross Bantam T51) leaves was examined. The inactive form of the enzyme and orthophosphate behaved kinetically as substrates for the reaction, which was catalyzed by an activating factor. This factor was bound by Blue Dextran Sepharose 4B and could be eluted by KCl at a concentration of 0.5m. The molecular weight of the maize leaf activating factor was about 88,000. Cibacron Blue 3G-A, a reactive moiety of Blue Dextran, inhibited the factor competitively with respect to the concentration of the inactive dikinase with a K(i) of 4.6 micromolar. Adenosine diphosphate and pyrophosphate were also found to be competitive inhibitors of activation, with respect to the inactive dikinase, giving K(i) values of 90 and 140 micromolar, respectively. Adenosine, other nucleotide diphosphates, and dinucleotides gave little or no inhibition of activation. These results suggest the association of a nucleotide, presumably nucleotide diphosphate, with the inactive form of pyruvate, orthophosphate dikinase.     

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

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

Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis

These studies provide information about the mechanism of the light/dark-mediated regulation of pyruvate, Pi dikinase (EC 2.7.9.1) in leaves. It is shown that inactivation is due to a phosphorylation of the enzyme from the beta-phosphate of ADP, and that activation occurs by phosphorolysis to remove the enzyme phosphate group. During ADP plus ATP-dependent inactivation of pyruvate, Pi dikinase in chloroplast extracts, 32P was incorporated into the enzyme from [beta-32P]ADP. Approximately 1 mol of phosphate was incorporated per mol of monomeric enzyme subunit inactivated. There was very little incorporation of label from ADP or ATP labeled variously in other positions with 32P or from the nucleotides labeled with 3H in the purine ring. Purified pyruvate, Pi dikinase was also labeled from [beta-32P]ADP during inactivation. In this system, phosphorylation of the enzyme required the addition of the "regulatory protein" shown previously to be essential for catalyzing inactivation and activation. During orthophosphate-dependent reactivation of pyruvate, Pi dikinase, it was shown that the enzyme loses 32P label and that pyrophosphate is produced. The significance of these findings in relation to regulation of the enzyme in vivo is discussed.     

Pyruvate orthophosphate dikinase from the immature grains of cereal grasses

Pyruvate orthophosphate dikinase has been identified in the green grains of eight cereal grasses, most of which are classified as C(3) plants. The wheat (Triticum aestivum L. cv. Lerma Rojo) grain enzyme was further investigated: activity was low in very young grains, increased to a maximum at about 25 days after anthesis, then returned to a low level as the grain matured. It appeared to be located in the aleurone layer. A procedure was developed for obtaining partially purified preparations of pyruvate orthophosphate dikinase from the ears of wheat, oat (Avena sativa L.), barley (Hordeum distichum L.), and rye (Secale cereale L.). These preparations were suitable for measuring activities in both the forward and reverse reaction directions. The affinities of these enzymes for the six substrates (pyruvate, orthophosphate, and ATP in the forward reaction; phosphoenolpyruvate, pyrophosphate, and AMP in the reverse reaction) were determined and found to be similar to the reported affinities of the enzyme from the leaves of the C(4) plant Zea mays. A possible role for pyruvate orthophosphate dikinase in cereal grains is considered briefly.     

Light and temperature dependence of the rate and degree of activation of pyruvate,Pi dikinase in vivo in maize

Activation of pyruvate,Pi dikinase by light was studied in leaf discs of maize which were illuminated for 1 h at light intensities ranging from approximately 3% to 50% of full sunlight and at temperatures of 10, 22.5, and 35°C. At the highest light intensity the degree of activation was similar and relatively independent of temperature between 10 and 35°C. Under low light the degree of activation was high at 10°C but decreased rapidly with increasing temperature. There was a similar effect of light and temperature on the activation of NADP-malate dehydrogenase.At low temperature, the rate of activation of pyruvate,Pi dikinase was relatively low and independent of the light intensity used and the rate of inactivation in the dark was extremely low. At high temperature, the rate of activation was high and dependent on the light intensity used while the rate of dark inactivation was also relatively high. The degree of activation is discussed in relation to the possible influence of light and temperature on the turnover between the active and inactive forms of pyruvate,Pi dikinase during illumination.This research was supported by the Japan-U.S. Cooperative Research Program (The Japan Society for the Promotion of Science, NFS Grant INT 78-17245), NSF Grant PCM 77-09284, by the Japanese Ministry of Education and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin.     

The effect of fructose 2,6-bisphosphate on the reverse reaction kinetics of fructose 1,6-bisphosphatase from bovine liver

The fructose 1,6-bisphosphatase reaction was investigated in the reverse direction by using fructose 2,6-bisphosphate. The effector was found to be a potent inhibitor of the reverse reaction substrates. Inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate was competitive, and slope replots were linear. In the context of other accumulated kinetic data, our results serve to support a Random Bi Uni mechanism as the most likely mechanism for the reverse reaction. In addition, two models consistent with the data are presented for the interaction of fructose 2,6-bisphosphate with fructose 1,6-bisphosphatase.     

Cold lability of pyruvate, orthophosphate dikinase in the maize leaf

Cold lability of pyruvate, orthophosphate dikinase was investigated using a homogeneous, purified enzyme preparation from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. Its stability was markedly reduced below about 10 C and the rate of cold inactivation followed first order kinetics at a concentration lower than about 0.1 milligram of enzyme per milliliter. Cold inactivation was little affected by pH in the range which gives good stability for the enzyme at warm temperatures and the enzyme activity was protected strongly by inclusion of substrates (pyruvate and phosphoenolpyruvate) and polyols such as sucrose, sorbitol, and glycerol. Loss of catalytic activity was accompanied by an apparent dissociation of a tetrameric form of the enzyme (9S form) into a new, more slowly sedimenting (5.1S) component. Inclusion of pyruvate at 4 mM in the cold-treated enzyme had no effect on the sedimentation value. A sharp change in activation energy of the dikinase-catalyzed reaction was observed near 12 C and its break point appears to be close to the generally accepted critical low temperature limit for the growth of maize plants.