By determining the 13C isotope effect on V/K with both a deuterated and an unlabeled substrate, and the deuterium isotope effect on V/K, it is possible to tell whether the 13C-sensitive and deuterium-sensitive steps are the same or not and, if they are different, to determine which comes first in the mechanism. If the two isotope-sensitive steps are the same, (1) deuteration increases the size of the observed 13C isotope effect, (2) narrow limits can be calculated for the intrinsic deuterium and 13C isotope effects, and (3) wider limits can be placed on the size of the commitments in the system. If an accurate determination of the tritium isotope effect on V/K is available, or if an a-secondary deuterium isotope effect which originates from the same step as the primary deuterium one and the corresponding 13C isotope effect with a-secondary deuterated substrate can be measured, an exact solution for the intrinsic isotope effects and the commitments is possible. With glucose-6-phosphate dehydrogenase, 13K, for label at C-1 of glucose is 0.9920, and 13C isotope effects are 1.0165, 1.03 16, and 1.01 76 with glucose 6-phosphate, glucose-I -d 6-phosphate, and glucose 6-phosphate (with TPN-4-4, respectively. The primary deuterium isotope effect on V/K of %e multistep nature of enzyme-catalyzed reactions usually decreases the magnitudes of observed isotope effects from the intrinsic isotope effects on the bond-breaking steps to somewhat lower values. Though this less than full expression can be a hindrance when trying to correlate isotope effects with transition-state structure, it can be quite useful in determining the relative rates of various steps and the sequence of these steps in a reaction mechanism. Often an isotopic substitution can be made which will affect the rate of only one particular step in a mechanism (typically via a primary deuterium isotope effect). By thus selectively changing the rate of this one step in the reaction mechanism and observing another isotope effect (either a deuterium or a heavy atom isotope effect) which is also expressed on a particular step of a mechanism, one can tell which isotope-sensitive step comes first in the mechanism or whether both isotope effects are on the same step. The concerted vs. stepwise controversy long associated with various enzymatic mechanisms can thus be unambiguously settled in many cases by application of the techniques which we will describe. We will show that the malic enzyme catalyzed oxidative decarboxylation of malate by triphosphopyridine nucleotide (TPN)' is a stepwise reaction with hydride transfer 2.97 and the a-secondary deuterium isotope effect of 1.00 allow calculation of intrinsic isotope effects and commitments in this system: Dk = 5.27, 13k = 1.0408, a-Dk = 1.054, cf = 0.75, and c, = 0.49. When deuterium-and 13C-sensitive steps are different, deuteration decreases the size of the observed I3C isotope effect. The data fit the equation [l3(V/K)H -I]/ [I3( V/K)D -11 = D(V/K)/DK, when the deuterium-sensitive step comes first but fit the equation...

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The carbon isotope effect at CO2 has been measured in the carboxylation of ribulose 1,5-bisphosphate by the ribulosebisphosphate carboxylase from Rhodospirillum rubrum. The isotope effect is obtained by comparing the isotopic composition of carbon 1 of the 3-phosphoglyceric acid formed in the reaction with that of the carbon dioxide source. A correction is made for carbon 1 of 3-phosphoglyceric acid which arises from carbon 3 of the starting ribulose bisphosphate. The isotope effect is k12/k13 = 1.0178 +/- 0.0008 at 25 degrees C, pH 7.8. This value is smaller than the corresponding value for the spinach enzyme. It appears that substrate addition with the R. rubrum enzyme is principally ordered, with ribulose bisphosphate binding first, whereas substrate addition is random with the spinach enzyme. The carboxylation step is partially rate limiting with both enzymes.

EMBL accession no. X52558 A 1.3 kb cDNA was isolated from a pea (Pisum sativum L.) cDNA library by immunoscreening with rabbit antiserum to spinach carbonic anhydrase (E.C. 4.2.1.1). The 984 bp open reading frame contained the entire coding region of the mature chloroplast protein and its transit peptide. The identity of the protein is verified by the strong homology with carbonic anhydrase from spinach (1). There is little sequence homology with mammalian isozymes (2). The transit peptide, based on Edman degradation of the mature polypeptide, is 104 amino acids. This is longer than commonly found for nuclear-encoded chloroplast proteins (3). Also unusual, even for the serineand theonine-rich chloroplast transit peptides, is a region of seven consecutive Ser residues bounded by Thr residues (amino acid residues 36-43). The molecular mass of the deduced 224 amino acid mature chloroplast polypeptide is 24.2 kd.

The labeling patterns in malic acid from dark (13)CO2 fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12-14 h dark (13)CO2 fixation the ratio of [4-(13)C] to [1-(13)C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The (13)C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87-89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following (13)CO2 fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [(13)C] malic acid ((13)CO2 or [1-(13)C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to (13)CO2 in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284-291). In the shortest times, only singly-labeled [4-(13)C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.

Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze-quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 ± 15 to 64 ± 10 nanomoles per milligram of chlorophyll [nmol/mg Chi]) while PEP levels increased (from 31 ± 4 to 68 ± 4 nmol/mg Chi, with a transient burst of 133 ± 16 nmol/mg Chi at 1 minute). Mesophyll cell levels of ATP increased (from 22 ± 4 to 48 ± 3 nmol/mg Chi) and ADP levels decreased (from 16 ± 4 to 7 ± 6 nmol/mg Chl) during the first minute of illumination. Upon darkening of the leaf and inactivation of dikinase, pyruvate levels initially increased in the mesophyll (from 160 ± 30 to a maximum of 625 ± 40 nmol/ mg Chi), and then slowly decreased to about the initial value in the light over an hour. PEP levels dropped (from 176 ± 5 to 47 ± 3 nmol/mg Chi) in the first 3 minutes and remained low for the remainder of the dark period. Mesophyll levels of ATP and ADP rapidly decreased and increased, respectively, about twofold upon darkening. The trends observed for these metabolite levels in the mesophyll cell during the light/dark regulation of pyruvate,orthophosphate dikinase activity suggest that pyruvate and PEP do not play a major role in vivo in regulating the extent of light activation (dephosphorylation) or dark inactivation (ADPdependent threonyl phosphorylation) of dikinase by its bifunctional regulatory protein. While the changes in ADP levels appear qualitatively consistent with a regulatory role for this metabolite in the light activation and dark inactivation of dikinase, they are not of a sufficient magnitude to account completely for the tenfold change in enzyme activity observed in vivo.Pyruvate,orthophosphate dikinase (EC 2.7.9.1) catalyzes the conversion of pyruvate, ATP and Pi to PEP,3 AMP, and

MATERIALS AND METHODSPhotosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO2 for growth, and a unique cotton cell line that grows at ambient CO2 (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C3 mesophyll leaf cells than the cell suspension cultures previously studied. The pattem of 14C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO2 fixation occurred primarily by the C3 pathway. Photorespiration occurred at 330 microliters per liter CO2, 21% 02 as indicated by the synthesis of high levels of 14C-labeled glycine and seine in a pulse-chase experiment and by oxygen inhibition of CO2 fixation. Short-term CO2 fixation in the presence and absence of carbonic anhydrase showed C02, not HC03-, to be the main source of inorganic carbon taken up by the low C02-requiring cotton cells. The cells did not have a C02-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low C02-requiring cotton cells, present in the high C02-requiring soybean cell lines, and absent in other high CO2 cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO2 Photoautotrophic Cell Suspension CulturesSoybean (Glycine max) cells and high C02-requiring cotton (Gossypium hirsutum) cells were grown at 5% C02 in a modified MS medium2 which contained thiamine and hormones as the only organic compounds (22). Cells were subcultured every 2 weeks. Low C02-requiring cotton cells were grown at ambient CO2, which was 660 ,L/L, in a modified MS medium (3,10), and were subcultured every 4 weeks. Cells were used in experiments 1 to 2 weeks after transfer, and were regularly found to be 90 to 95% viable as determined by phenosafranine staining (30). Prior to use, the cells were washed three times with the appropriate buffer (usually 30 mm Mops, pH 7.0). Cells stored for 4 h in buffer at 11 sE/m2 *s in a test tube or Petri dish showed less than a 10% decrease in the rate of '4C02-fixation. CO2 FixationCells (10-20 ,ug

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