Malate inhibition of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate²⁻ concentration. Of the reaction components only HCO₃⁻ increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K_m, but no effect on V_max. Enzyme preincubated with malate shows the same effect of malate on K_m, but in addition V_max is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.     

Limited proteolysis by trypsin influences activity of maize phosphoenolpyruvate carboxylase

Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by very low concentrations of trypsin at 37 degrees C. PEP+Mg2+ and several other effectors of PEP carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition (in presence of PEP, Mg2+ and HCO3) although yielded a truncated subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic condition, only malate sensitivity was appreciably affected. Significant protection of the enzyme activity against trypsin during catalytic phase could be either due to a conformational change induced on substrate binding. Several lines of evidence indicate that the inactivation caused by a cleavage at a highly conserved C-terminal end of the subunit.     

Malate-Induced Hysteresis of Phosphoenolpyruvate Carboxylase from Crassula argentea

The hysteretic behavior of phosphoenolpyruvate (PEP) carboxylase from Crassula argentea has been investigated. Incubation of the purified enzyme with the inhibitor malate prior to starting the reaction by the addition of PEP resulted in a kinetic lag of several minutes duration. The length of the lag was inversely proportional to the enzyme concentration, suggesting subunit association-dissociation as the hysteretic mechanism, rather than a mechanism based on a slow conformational change in the enzyme. Dynamic laser light scattering measurements also support this conclusion, showing that the diffusion coefficient of malate-incubated enzyme slowly decreased after the reaction was started by the addition of PEP. Lags were observed only at pH values of 7.5 or lower. Maximum lags were observed after 10 min of preincubation with malate. Fumarate and succinate, which like malate caused mixed inhibition, also caused lags. In contrast, no lag was induced by malate in the presence of PEP or by the competitive inhibitor phosphoglycolate. The activators glucose 6-phosphate and malonate decreased the malate-induced lag.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at −70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage—which is composed of an equilibrium mixture of dimer and tetramer—with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open.     

Effects of fusicoccin on the activity of a key pH-stat enzyme,PEP-carboxylase

The phytotoxin fusicoccin (FC) causes rapid synthesis of malate in coleoptile tissues, presumably via phosphoenolpyruvate (PEP) carboxylase coupled with malate dehydrogenase. The possibility that FC directly affects PEP carboxylase in Avena sativa L. and Zea mays L. coleoptiles was studied and rejected. The activity of this enzyme is unaffected by FC whether FC is added in vitro or a pretreatment to the live material. FC does not change the sensitivity of the enzyme to bicarbonate or malate. The activity of FC, instead, appears to be indirect. The pH sensitivity of PEP carboxylase is such that its activity, and thus the rate of malate synthesis, may be enhanced by an increase in cytoplasmic pH accompanying FC-induced H⁺ excretion. Since the enzyme is also particularily sensitive to bicarbonate levels, malate synthesis may also be enhanced by FC-induced uptake or generation of CO₂.     

Maize leaf phosphoenolpyruvate carboxylase : oligomeric state and activity in the presence of glycerol

Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H⁺ concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S_0.5) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S_0.5 in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase.     

Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants

The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C₄ plant, exhibited a temperature optimum of 35–37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17–20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C₄ plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C₄ acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C₄ acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C₄ enzyme while its activators showed no significant effect. PEP carboxylase from C₃ species was not protected from thermal inactivation by the C₄ acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C₄ plants.Abbreviations CAM crassulaccan acid metabolism - Chl chlorophyll - Ea activation energy - PEP phosphoenolypyruvate Journal Series Paper, New Jersey Agricultural Experiment Station     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

The role of oligomerization in regulation of maize phosphoenolpyruvate carboxylase activity. Influence of Mg-PEP and malate on the oligomeric equilibrium of PEP carboxylase

A purification procedure which yields a near homogenous preparation of phosphoenolpyruvate (PEP) carboxylase from the leaves of Zea mays is reported. The enzyme had a final specific activity of 33.3 micromoles per minute per milligram protein. Size exclusion high performance liquid chromatography and dynamic laser-light scattering spectroscopy showed that PEP carboxylase exists in an equilibrium of aggregates. Enzyme predominantly in the dimeric configuration is less active (when assayed at sub-optimal Mg-PEP concentrations, less than 0.4 millimolar) than when in its tetrameric arrangement. The difference in activity diminishes and disappears as the concentration of the substrate Mg-PEP increases. The substrate drives the equilibrium toward the tetramer, while malate, an inhibitor of PEP carboxylase, shifts the equilibrium toward the dimer. It thus appears that the quaternary structure (oligomeric state) of maize PEP carboxylase can be regulated by the naturally occurring effector molecules Mg-PEP and malate which in turn can control the enzyme's activity.     

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone     

Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

Modification of cytokinins by cauliflower microsomal enzymes

Two homozygous mutant lines of barley (Hordeum vulgare L.) R3202 (Lt1b/Lt1b) and R3004 (Lt2/Lt2), are resistant to lysine plus threonine. They contain aspartate kinase isoenzymes with lost or decreased feedback sensitivity to lysine in either isoenzyme AKII (R3202) or isoenzyme AKIII (R3004). A homozygous double mutant line (Lt1b/Lt1b, Lt2/Lt2) has now been constructed that grows vigorously on 8 millimolar lysine, 8 millimolar threonine, and 1 millimolar arginine. Both AKII and AKIII from the double mutant have altered lysine sensitivities, identical to those previously observed in R3202 and R3004, respectively. Aspartate kinase activity in extracts of leaves, roots, and the maturing endosperm of the double mutant was much less sensitive to lysine inhibition than the enzyme in comparable extracts of the parent cv Bomi, suggesting that aspartate kinase is expressed in a similar manner in different tissues of barley.

A further mutant, R2501, resistant to lysine plus threonine has now given rise to a homozygous line (Lt1a/Lt1a), which had previously not been possible. AKII isolated from the homozygous line was completely insensitive to 10 millimolar lysine; however, the combined action of 10 millimolar lysine and 0.8 millimolar S-adenosylmethionine inhibited it by 60%, demonstrating the retention of some of the regulatory characteristics of the wild type enzyme.

     

Inhibition of phosphoenolpyruvate carboxylase by malate

Malate has been noted to be a `mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses

Phosphoenolpyruvate (PEP) carboxylase activity in immature `Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.     

Oligomerization and regulation of higher plant phosphoenolpyruvate carboxylase

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K_m, and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.     

Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase

Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/1) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^?) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from hight Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.