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R T Wedding

Publications and source records attributed to R T Wedding.

At least 37 records · Page 2Linked to original sources
PubMed1988

Role of Magnesium in the Binding of Substrate and Effectors to Phosphoenolpyruvate Carboxylase from a CAM Plant.

The binding of phosphoenolpyruvate, malate, and glucose 6-phosphate to phosphoenolpyruvate carboxylase purified from Crassula argentea Thunb. was measured using both the intrinsic tryptophan fluorescence of the enzyme and the extrinsic fluorescence of the complex of 8-anilino-1-napthalenesulfonate with the enzyme. It was found that the substrate phosphoenolpyruvate can bind in the absence of magnesium but is bound in greater quantities and more tightly when magnesium is present. Malate reduces the binding of phosphoenolpyruvate, while glucose 6-phosphate increases the binding of the substrate. Glucose 6-phosphate requires magnesium to bind to the enzyme, while malate does not. The general trends from the binding experiments using fluorescence methods were confirmed by activity determinations using assays performed in the absence of magnesium.

Journal Article↗
PubMed1988

Kinetic studies of the form of substrate bound by phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase isolated from maize (Zea mays L.) leaves was assayed with varying concentrations of free phosphoenolpyruvate at several fixed-varying concentrations of free magnesium higher than required to saturate the enzyme reaction. These assays produced velocity data which were found to form a family of individual lines when plotted against free phosphoenolpyruvate or against total phosphoenolpyruvate, but not when plotted against the concentration of the complex of phosphoenolpyruvate with magnesium. In this latter case, the points from all the fixed-varying concentrations fell on the same line, which can be fitted to a modified Michaelis-Menten equation with a multiple correlation coefficient R(2) = 0.995. Similar results were obtained when the enzyme from the C(4) plant maize was assayed with manganese rather than magnesium and when phosphoenolpyruvate carboxylase from leaves of the C(3) plant wheat (Triticum vulgare Vill.) was assayed with magnesium. However, at pH 7.0 the enzyme from the Crassulacean acid metabolism plant Crassula argentea did not produce a satisfactory single line when plotted against the complex of metal ion and substrate, but did so when the assay pH was raised to 8.0. It is concluded that in general the preferred form of substrate for phosphoenolpyruvate carboxylase is the complex of phosphoenolpyruvate with the metal ion.

Journal Article↗
PubMed1987

Evidence for a multiple subunit composition of plant NAD malic enzyme.

Malate dehydrogenase (decarboxylating) (EC 1.1.1.39) was purified to near homogeneity from both a C3 plant, Solanum tuberosum, and a CAM plant, Crassula argentea. Sodium dodecyl sulfate-gel electrophoresis of both enzymes revealed an alpha,beta subunit composition with corresponding molecular mass assignments of 61,000 and 55,000 daltons. Isoelectric focusing under native conditions showed only one constituent malic enzyme form with an isoelectric point of 5.1. No evidence of additional isoenzymes was found. Urea isoelectric focusing showed the alpha subunit to be more acidic than the beta subunit. Peptide mapping by limited proteolysis with Staphylococcus aureus V-8 protease, trypsin, and endoproteinase Arg-C eliminated the possibility that a precursor-product relationship may have existed between the two subunits and demonstrated that they each possess unique primary sequences. Further support for this conclusion was obtained when significant differences in the contents of glutamic acid, isoleucine, and arginine were revealed by amino acid analysis of the isolated subunits. There was no apparent activity associated with the separated subunits (as resolved by urea-DEAE chromatography), but activity could be found in a reconstituted system, thereby indicating an (alpha,beta)n protomeric configuration. This is the first case where malic enzyme has been conclusively shown to be constructed from nonidentical subunits. This phenomenon has been observed only for the NAD malic enzyme isolated from plants.

Amino Acids↗
PubMed1987

Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula.

The 13C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on Vmax. This indicates a stepwise conversion of malate to pyruvate and CO2 with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean acid metabolism while maintaining the catalytic events found in malic enzymes from animal sources.

Carbon Isotopes↗
PubMed1987

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 degrees 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.

Journal Article↗
PubMed1987

pH Effects on the Activity and Regulation of the NAD Malic Enzyme.

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg(2+) and NAD(+) but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate(-2). This is characteristic of an induced conformational change. The slope of the V(max) or V(max)/K(m) profiles is steeper on the alkaline side of the pH optimum. The K(m) for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K(m pH6.7) = 9.2 millimolar, K(m pH7.7) = 28.3 millimolar). Similar changes in K(m) have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V(max)/K(m) profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.

Journal Article↗
PubMed1987

Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases.

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C(4) enzyme to inhibition by malate is also unaffected by preincubation with these ligands.

Journal Article↗
PubMed1987

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study.

The effect of temperature in the range from 10 to 35 degrees C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C(4) plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V(max) for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C(4) enzyme has an inflection at 27 degrees C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K(m) for the two enzymes reveals the startling fact that at temperatures above 20 degrees C an increasing temperature causes an increase in K(mPEP) for the CAM enzyme while the C(4) enzyme displays a decreased K(m) as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15 degrees C to 70% at 35 degrees C. For the C(4) enzyme the percent inhibition drops from about 60% at 20 degrees C to 2% at 30 degrees C. Similar opposite behavior of the two enzymes is found with the K(i) for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25 degrees C.

Journal Article↗
PubMed1986

Regulation of the NAD Malic Enzyme from Crassula.

Using size exclusion chromatography, the nicotinamide adenine dinucleotide malic enzyme purified to near homogeneity from leaves of Crassula argentea was found to exist in at least three aggregational states (dimer, tetramer, and octamer). These forms differ in their apparent kinetic characteristics in initial rate assays, but all display similar characteristics at the steady state. The presence of 50 millimolar malate during chromatography causes a shift in favor of the smaller forms with the tetramer predominating. The native enzyme, when diluted 1/1000 and incubated 18 hours in buffer of high ionic strength, changes its steady state kinetic parameters to ones which indicate a low activity and low affinity for malate. When 50 millimolar malate or 50 micromolar coenzyme A are present the loss of activity and increase in K(m) is reduced. When both malate and coenzyme A are present the effects in minimizing the change in kinetic characteristics are additive.

Journal Article↗
PubMed1986

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 malate(2-) concentration. Of the reaction components only HCO(3) (-) 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.

Journal Article↗
PubMed1985

Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms.

Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.

Carboxy-Lyases↗
PubMed1985

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula.

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K(m) for phosphoenolpyruvate and reducing V(max). With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K(m) for phosphoenolpyruvate, but having little effect on V(max). The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K(m) for phosphoenolpyruvate and an increased V(max), but the night, or insensitive, form shows only an increase in V(max) in response to glucose-6-phosphate.

Journal Article↗
PubMed1984

Modulation of the activity of NAD malic enzyme from solanum tuberosum by changes in oligomeric state.

The effects of pH, NaCl, and malate2- on the equilibrium between dimeric and higher-molecular-weight forms of NAD malic enzyme from Solanum tuberosum var. Chieftain have been analyzed by monitoring the kinetic changes associated with disaggregation [S. D. Grover and R. T. Wedding (1982) Plant Physiol. 70, 1169-1172]. At pH values above 7.0 the enzyme was disaggregated to the dimeric, high-Km(malate) form by preincubation with NaCl, with a half-maximal effect at 25 mM. At low pH the enzyme remained in the low-Km(malate) (tetramer or octamer) form. Malate protected against disaggregation to the high-Km form in preincubation, and this effect was half-maximal at 6 mM. At pH 7.3, in the absence of malate, half-maximal disaggregation occurred at 580 nM enzyme. Varying the enzyme concentration in the assay led to kinetic changes which fit equations based on an associating enzyme model [B. I. Kurganov (1967) Mol. Biol. (Moscow) 1, 17-27]. This analysis confirmed that the dimer has intrinsic activity, with Vm somewhat lower than that of the tetramer but a Km(malate) that was 9-fold higher than that of the tetramer. Malate decreased the Kd for disaggregation of the enzyme during assay approximately 20-fold, with a half-maximal effect at 3 to 4 mM. In contrast, high NaCl concentrations in the assay increased the Kd for disaggregation in a manner which was competitive with the effect of malate on Kd. The physiological significance of these aggregation state changes is discussed.

Hydrogen-Ion Concentration↗
PubMed1984

Kinetic properties of NAD malic enzyme from cauliflower.

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD+ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD+ and malate2- at a saturating concentration of Mg2+ or by varying Mg2+ and NAD+ at a saturating level of malate2-are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate2- in the absence of either Mg2+ or NAD+. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg2+, the enzyme can also use Mn2+ and Co2+. Using Co2+ in place of Mg2+ does not change Vmax or Km, malate2- but the Km for metal and NAD+ are greatly decreased. At pH 7.0 and above, Mn2+ isotherms and malate2- curves with Mn2+ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.

Ethylmaleimide↗
PubMed1983

Allosteric regulation of the NAD malic enzyme from cauliflower: activation by sulfate.

Activation of the NAD malic enzyme by sulfate has been found to be due only to free, uncomplexed SO42-; the complex of sulfate with divalent cations has no measurable effect on the enzyme. Activation by SO42- is shown to result from a decrease in the Km for malate2-. Thus, activation is observed only at less than saturating levels of this substrate. The interactions of NAD+ and Mg2+ with the enzyme are not affected by SO42-. Response of the activity of the enzyme to SO42- is biphasic in that the activation seen at low SO42- concentrations is overcome as the level of the effector is increased so that at very high SO42- concentrations, activation disappears. This deactivation process is not simply a reversal of the activation mechanism; instead, it involves a decrease in the intrinsic Vmax of the reaction. The response to SO42- is also affected by the presence of other anionic effectors of the malic enzyme. Fumarate2- and phosphate are shown to directly affect the activation process by increasing the affinity of the enzyme for SO42-. While Cl- does not greatly affect the extent of stimulation, it does inhibit the enzyme without reducing the activated rate so that the apparent percentage activation over the control is very large, due to the lowered control rate. In contrast to the sensitivity of the malic enzyme reaction to pH, activation by SO42- appears to be independent of H+ concentration. The possibility that sulfate is a physiological effector of this and other plant mitochondrial enzymes is discussed.

Allosteric Regulation↗
PubMed1983

Allosteric regulation of the NAD malic enzyme from cauliflower: activation by fumarate and coenzyme A.

Activation of the NAD malic enzyme is shown to be caused by free, uncomplexed fumarate2-. Mg-fumarate has no detectable effect on the enzyme. Fumarate2- isotherms are biphasic in that they consist of an activating as well as a deactivating region. Activation is shown to result from an increase in the affinity of the enzyme for malate2- while deactivation results from a reduction in Vmax. Phosphate does not affect the response of the enzyme to fumarate2-, while Cl- inhibits the enzyme in a manner that cannot be overcome by fumarate2-. SO42-, another activator of the malic enzyme, reduces the Ka for fumarate2- from 3.9 to 2.1 mM. Activation of the enzyme by coenzyme A (CoA) is hyperbolic with a Ka for CoA of 2.1 microM. Fumarate2- reduces this value to 1.2 microM. CoA, like SO42-, is able to increase the affinity of the enzyme for fumarate2-, decreasing its Ka by 56%. An additional effect of fumarate2- is to cause the interconversion of different catalytic forms of the enzyme which exist when Mg2- is limiting. On the basis of these results, a model of the number and types of allosteric sites present on the NAD malic enzyme is proposed.

Allosteric Regulation↗
PubMed1983

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea.

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.The enzyme uses either Mg(2+) or Mn(2+) as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn(2+) the K(m) for malate(2-) is lower than with Mg(2+), but V(max) is lower than with Mg(2+). In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C(3) plants. In the reverse reaction, run with Mn(2+), the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 x 10(-3) molar.The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.The enzyme is activated by coenzyme A with K(a) = 4 micromolar. AMP also shows competitive activation, with K(a) = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate(2-) is a strong activator, with K(a) = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K(i) = 4.9 millimolar.

Journal Article↗