oxygenase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 2, June, pp. 610-617, 1983

Pyridoxal Phosphate as a Probe in the Active Site of Ribulose Bisphosphate Carboxylase/Oxygenase ANIL

S. BHAGWATi

AND

BRUCE A. MCFADDEN’

Biochemistry/Biophysics Program and Institute of Biological Chemistry, Washington State University, Pullman, Washington 99X$&30 Received December 2, 1982, and in revised form January

31, 1983

Ribulose bisphosphate (RuBP) carboxylase is rapidly and irreversibly inactivated by photooxidation sensitized by pyridoxal phosphate. Both pyridoxal and pyridoxamine phosphate were much less effective in sensitizing the photooxidation even when used at twice the concentration of pyridoxal phosphate. These results imply that pyridoxal phosphate binds at the active site not only through a Schiff base, but also through ionic interaction with the phosphate binding region. Spectral analysis of the photooxidized enzyme showed a new absorption maximum at 325 nm due to reduction of the Schiff base between pyridoxal phosphate and a lysyl residue with concomitant oxidation of a histidine residue. The stoichiometry of photooxidative [3H]pyridoxal phosphate incorporation was 0.87 mol/mol of a 70,000-dalton large subunit-small subunit combination. Studies with 3H-labeled diethyl pyrocarbonate showed that both photooxidation and carbethoxylation occur at the same histidine residue. However, photooxidation by pyridoxal phosphate is very specific for an active site histidine residue due to the high specificity of this affinity label. Several competitive inhibitors with respect to ribulose bisphosphate offered appreciable protection against pyridoxal phosphateinduced photooxidation of the enzyme. The photooxidized enzyme showed an increase in the net negative charge on the protein which was evident from the higher mobility of the photooxidized enzyme toward the anode in polyacrylamide gel electrophoresis.

Ribulose l,&bisphosphate (RuBP)~ carboxylase from several sources has been shown to be completely inhibited by pyridoxal phosphate (l-4). The inhibition results from the reversible binding of pyridoxal phosphate to a lysine residue of the enzyme. The inhibition is reversible upon dilution of the enzyme. Pyridoxal phosphate bound to a specific lysyl residue has been shown to sensitize photooxidation of

an adjacent essential histidine residue in the case of several enzymes (5-10). Thus pyridoxal phosphate has been used in two different ways to identify critical amino acid residues in proteins: (1) as a reagent for identifying essential lysyl residues; (2) as a photosensitizer for the oxidation of a neighboring histidine. It has been shown that lysyl residues play an important role in the catalytic mechanism of RuBP carboxylase. There are eight essential lysine residues in the 560,000-dalton higher plant enzyme comprising eight large 55,000-dalton and eight small 15,000-dalton subunits. The essential lysine residues are located on large subunits (4). It has also been established that 8 more nonessential lysyl residues are located outside the active site and that they

i On leave from Biology and Agriculture Division of Bhabha Atomic Research Centre, Bombay, India. *Author to whom correspondence should be addressed. ’ Abbreviations used: RuBP, ribulose 1,5-bisphosphate; Bicine, N,N-bis(2-hydroxyethyl)glycine; DTT, dithiothreitol; PLP, pyridoxal phosphate; Mops, 4morpholinopropanesulfonic acid; DEP, diethyl pyrocarbonate. 0003-9861/83 $3.00 Copyright All rights

8 1983 by Academic Press, Inc. of reproduction in any form reserved

610

ACTIVE

SITE

OF RIBULOSE

react much more slowly with pyridoxal phosphate (4). Our studies (11-13) have shown that one histidyl residue/large subunit is essential for activity and we have postulated a role for this residue in catalysis (14-15). The exact location of this histidine residue in the primary structure of the enzyme is not known, and in the present work pyridoxal phosphate has been used to probe for an essential histidyl residue adjacent to the essential lysyl residue. An attempt has also been made to ascertain whether the histidine residue modified by photooxidation is the same as that modified by another histidine selective reagent, diethyl pyrocarbonate, used in our earlier studies (11-13). We now describe evidence that suggests the presence of proximal lysyl and histidyl residues at the active site of RuBP carboxylase from spinach. MATERIALS

AND

METHODS

RuBP carboxylase was purified to homogeneity from spinach leaves (final specific activity: 2.2 units/mg) according to the method of Berhow et al. (16). [3H]Pyridoxal phosphate was prepared according to the method of Stock et al. (17). Pyridoxal phosphate, pyridoxal-HCl, pyridoxamine phosphate, and diethylpyrocarbonate were purchased from Sigma Chemical Company. All other chemicals were of analytical grade. Light intensity was measured using a Lambda L-1-185 Quantum/ Radiometer/Photometer. Prior to each experiment, the enzyme, stored as a 55% ammonium sulfate suspension, was centrifuged, dissolved in 100 mM Bicine (pH 7.6), and passed through a column of Sephadex PD-10 to remove ammonium sulfate. The enzyme was fully activated with 10 mM NaHCO,, 20 mM MgCle, and 2 mM dithiothreitol (DTT) at 30°C for 15 min. Procedure for photooxidation The apparatus for photooxidation was a water-jacketed cell usually used with a Gilson Oxygraph. Pyridoxal phosphate at a final concentration of 1 mM (unless otherwise specified) was added in the dark to the enzyme solution in 100 mM Bicine, pH 7.6, containing 10 mM NaHCOa, 20 mM MgCla, and 2 mM DTT. Dithiothreitol was routinely used in the photooxidation experiments since this reagent quantitatively protected protein sulfhydry1 groups against oxidation (unpublished observation). For kinetic experiments, the protein concentration was 0.8-l mg/ml in a volume of 2 ml. The sample was divided into duplicate pairs, and one pair

BISPHOSPHATE

CARBOXYLASE

611

was kept in the dark and the other pair exposed to light. A 20-~1 portion was withdrawn from the dark control and from the illuminated sample at desired time intervals. The reactions were either stopped by addition of NH,OH to a final concentration of 10 mM or by 20-fold dilution with the assay buffer. Photooxidation experiments were carried out at 25°C and samples were illuminated with an air-cooled projector 300-W lamp at a distance of 7 cm yielding a light intensity of 3 X lo5 lux at the outside edge of the tube. Pyridoxal phosphate was removed by gel filtration before spectral studies. Reaction with diethyl pyrocarbonate. Modification studies with diethyl pyrocarbonate were done as described previously (12,13). Unreacted pyridoxal phosphate was removed and modification by [aH]diethyl pyrocarbonate was conducted for 15 min at a concentration of 1 m&f. Excess reagent was removed by extensive dialysis against 67 mM sodium phosphate, pH 7.6, at 4°C. The degree of carbethoxylation by diethyl pyrocarbonate was determined spectrophotometrically using a molar extinction coefficient of 3200 Me' cm-’ (18). In protection experiments, the photooxidized enzyme was preincubated with competitive inhibitors at the concentrations indicated in the Tables for at least 10 min at 30°C prior to addition of [aH]diethyl pyrocarbonate or photooxidation by pyridoxal phosphate. Radioactivity was counted in glass vials using a liquid scintillation spectrometer. Assay for carboxylase activity. RuBP carboxylase activity was assayed according to the general method of McFadden et al. (19). Polyacrylamide gel electrophoresis was done according to the method of Davis (20). Fluorescence emission was recorded in a PerkinElmer MPF-3L spectrofluorometer. The excitation and emission spectra indicated maxima at 286 (excitation) and 336 nm (emission). At these wavelengths, all measurements were carried out at 0.1 mg/ml enzyme concentration. Sulfhydryl grump analysis. Sulfhydryl analysis using 5,5’-dithiobis(2-nitrobenzoate) were done according to the method of Ellman (21). Spectrophotometric measurements were done in a Cary-14 recording spectrophotometer. RESULTS

Effect of photooxidation on enzyme activity. In normal laboratory light the inhibition of RuBP carboxylase by pyridoxal phosphate is reversible either by dilution, dialysis, gel filtration, or treatment with NHzOH. An irreversible inactivation occurs if the enzyme is exposed to light in the presence of this reagent. The process of inactivation follows pseudo first-order

612

BHAGWAT

AND

reaction kinetics. (Fig. 1). The rate of inactivation depends on the concentration of pyridoxal phosphate. A plot tl12 vs l/PLP (Fig. 1, inset) is linear and yields a y intercept equal to the minimum t112 of 10 min. Thus the inactivation shows saturation kinetics with respect to pyridoxal phosphate. The enzyme undergoes no irreversible loss of enzymatic activity when illuminated for the same amount of time in the absence of pyridoxal phosphate. The degree of photooxidation was also dependent on the protein concentration during irradiation (Table I). At the lowest protein concentration more than 80% loss of activity was observed, whereas at the highest concentration (2 mg/ml) about 50% inactivation was seen after 20 min of irradiation. Presumably inactivation of RuBP carboxylase as described did not result from the oxidation of other residues like Cys, Tyr or Met because the enzyme was protected by DTT during photooxi-

.(C IO

20

30

40

TIME OF IRRADIATION (mid of inactivation of ribulose l,Sbisphosphate carboxylase by photooxidation. The reaction conditions are described under Materials and Methods. The concentrations of pyridoxal phosphate (PLP) during irradiation were 0.1 mM (O), 0.25 mM (0), 0.5 mM (A), and 1 mM (A). The inset shows a plot of inactivation half-time, &, versus l/[PLP]. FIG. 1. Kinetics

MC FADDEN TABLE PHOTOINACTIVATION AS A FUNCTION

OF SPINACH RuBP CARBOX~LASE OF ENZYME CONCENTRATION”

RuBP carboxylase bdml) 0.2 0.5 1 2

I

7%of original activity 16 24 33 49

’ RuBP carboxylase from spinach was photoinactivated for 20 min at 1 mM pyridoxal phosphate and the enzyme concentrations indicated. All other ronditions were as in Fig. 1.

was protected by DTT during photooxidation, and before assay was again treated with DTT. Moreover, the absorbance at 280 nm and thiol titer were not altered by photooxidation of the enzyme, ruling out the oxidation of Tyr and Cys residues, respectively. Photooxidation in the presence of pym’doxal and pyridoxamine phosphate. In order to investigate the specificity of photooxidation by pyridoxal phosphate, we have carried out photooxidation experiments using pyridoxal instead of its phosphorylated derivative and pyridoxamine phosphate. Both of these compounds are much less effective than pyridoxal phosphate (Fig. 2). Photooxidation in the presence of competitive inhibitors. In order to check whether the site of photooxidation is located at or near the active site, the extent of photooxidation of the enzyme was determined in the presence and absence of several competitive inhibitors with respect to RuBP. The enzyme was preincubated with the inhibitors at the indicated concentrations for 10 min before pyridoxal phosphate-sensitized photooxidation. The results presented in Table II show that the competitive inhibitors tested protected against photooxidation of the enzyme. The carryover of each inhibitor to the assay mixture was minimized by dilution and was noninhibitory at the saturation concentration of RuBP employed. Polyacrylamide gel analysis of the pho-

ACTIVE

SITE

OF RIBULOSE

A Pyridoxamine-

155 0

IO

20

30

P

40

TIME OF IRRADIATION (min) FIG. 2. Kinetics of inactivation of RuBP carboxylase by photooxidation in the presence of 1 mM pyridoxal phosphate (PL-P), 2 mM pyridoxal (PL), and 2 mM pyridoxamine phosphate. Other reaction conditions were the same as in Fig. 1.

tooxidized enzyme. The enzyme kept in the dark or photooxidized at 1 mM pyridoxal phosphate for various times was subjected to electrophoresis in a slab gel polymerized from 5% acrylamide. The enzyme photooxidized for 30 or 50 min showed faster mobility toward the anode than that representing the dark control (Fig. 3). This presumably reflected the incorporation of the pyridoxal phosphate moiety with a net negative charge. Photooxidative incorporation of [3H]pyridoxal phosphate. Progressive incorporation of [3H]pyridoxal phosphate into the enzyme occurred as a function of time of irradiation (Table III). The incorporation was not altered by treatment with hydroxylamine followed by extensive dialysis (Table III), corroborating the conclusion from gel electrophoretic studies that the pyridoxal phosphate moiety had been irreversibly bound. Moreover, the incorporation of tritium (last entry, Table III) corresponded to 0.87 mol/mol of large and small subunit combination at complete inactivation.

BISPHOSPHATE

613

CARBOXYLASE

Spectral analysis of the photooxidized enzyme. Pyridoxal phosphate shows a characteristic absorption maximum at 338 nm. The binding of pyridoxal phosphate to lysine residues results in formation of a Schiff base having an absorption maximum at 432 nm. The reduction of this base with borohydride results in the formation of new absorption peak at 325 nm and loss of the peak at 432 nm (4). Ribulose bisphosphate carboxylase photooxidized with pyridoxal phosphate showed a broad absorption band centered around 325 nm (Fig. 4). Several other enzymes photooxidized with pyridoxal phosphate have shown similar absorption characteristics (8-10). The general interpretation has been that a nearby photooxidized histidyl residue is involved in the reduction of the Schiff base. To test this possibility, enzyme was modified with diethyl pyrocarbonate and then subjected to illumination in the presence of pyridoxal phosphate. This treatment abolished the characteristic absorption maximum at 325 nm (Fig. 5). Fluorescence spectra of the photooxidized enzyme showed maxima for excitation at 286 nm and emission at 336 nm.

TABLE

II

PROTEC~IONAGAINSTPYRIDOXALPHOSPHATEINDUCEDPHOTOOX~DATIONBY COMPETITIVE INHIBITORS~

Effector None Sedoheptulose 1,‘7-bisphosphate 2-Carboxy-D-mannitol 1,6-bisphosphate Fructose 1,6-bisphosphate 6-Phospho-n-gluconate

Concn (n-f)

7%of control activity 36

2

74

1 2 2

6’7 74 47

a The enzyme at 2 mg/ml was irradiated for 40 min at 1 mM pyridoxal phosphate in the presence or absence of indicated effecters. The enzyme was preincubated with each effector for 10 min before irradiation. The carboxylase activity was measured after suitable dilution of the enzyme subsequent to photooxidation. The dark control contained an equivalent amount of the competitive inhibitor that was carried over during assay.

614

BHAGWAT

AND

The fluorescence yield was not appreciably lower during the first 20 min of photooxidation as compared to the dark control. However, beyond 20 min, the photooxidized enzyme gave an appreciably lower fluorescence yield.

The nature of the histidine residue modified by diethyl pyrocarbonate and photooxidation. In order to determine whether the same histidine residue is modified by photooxidation and diethyl pyrocarbonate, the enzyme was first photooxidized with pyridoxal phosphate to various extents of inactivation. After removal of excess pyri-

0

m v FIG. 3. Slab gels polymerized from 5% acrylamide after electrophoresis of the control and photooxidized RuBP carboxylase. The enzyme at 0.5 mg/ml was photooxidized at 1 mM pyridoxal phosphate for 30 and 50 min. A duplicate set was kept in dark for the same time. All samples were incubated with 2 mM DTT during and after photooxidation. Protein (10 pg) was loaded in each slot. The migration was toward anode (bottom of the gel). Lanes contained from left to right: 1 and 2, enzyme photooxidized for 50 min; 3 and 4, enzyme photooxidized for 30 min; 5 and 6, dark control (50 mink 7 and 8, control not treated with pyridoxal phosphate.

MC FADDEN TABLE

III

INCORPORATIONOF[~H]PYRIDOXAL PHOSPHATE DURING PHOTOOXIDATION" Time of irradiation (min) 10 20 30 40

% of inactivationb 21 40 50 60

mol [3H]PLP/mol of subunit” 0.20 0.30 0.35 0.52d

“The enzyme at 2.0 mg/ml was irradiated at 0.4 mM [3H]pyridoxal phosphate (PLP) for the indicated time. All other conditions were as described under Materials and Methods. An aliquot was withdrawn for activity measurement. The remaining portion was treated with 10 mM NHzOH and was dialyzed extensively against 4000 vol 67 mM Na phosphate buffer, pH 7.6, at 2°C. The activity irreversibly bound to the enzyme was counted in a liquid scintillation spectrometer. b The enzyme kept in the dark for the same amount of time at 0.4 mM rH]pyridoxal phosphate and treated identically served as a control for nonspecific binding of PLP to the enzyme. “A 70,000-dalton combination of large and small subunit. d This reflected the incorporation of 4310 cpm minus 862 cpm which had been incorporated in the corresponding control.

doxal phosphate, the protein was treated with [3H]diethyl pyrocarbonate as described under Materials and Methods. The results presented in Table IV show that, even after 90% loss of activity due to photooxidation, ca. 9 histidines could be modified by diethyl pyrocarbonate per enzyme molecule. A plot of the decay of activity vs moles histidine modified per mole of enzyme is linear with correlation coefficient (in linear regression analysis) of 0.96 and extrapolates to a value of 8 at zero percent residual activity. It may be mentioned here that previous studies (12, 13) have shown that there are two modifiable histidines per 70,000-dalton combination of large and small subunits or 16 residues/mol of enzyme. However, only one residue is essential (13). The results (Table IV) suggest, then, that bound pyridoxal phosphate, being an active site-directed reagent, photooxidizes the essential histidine and that

ACTIVE

0”

300

320

1

SITE

340

WAVELENGTH

1

OF RIBULOSE

360

1

1 360

’

BISPHOSPHATE

CARBOXYLASE

peating unit. Thus all evidence suggests that the site of both photooxidation and modification by diethyl pyrocarbonate is the same histidine residue located at the active site of RuBP carboxylase/oxygenase. It was impossible to probe the photooxidative modification of histidyl residues by amino acid analysis of hydrolysates because spinach RuBP carboxylase contains 139 histidines/mol (24). The loss of eight residues could not be reliably estimated.

(nm)

FIG. 4. Absorption spectrum of photooxidized RuBP carboxylase. Enzyme at 1 mg/ml was photooxidized for 30 min as described under Materials and Methods. After removing excess pyridoxal phosphate from the dark control and photooxidized enzyme, the spectra were run in a Cary 14 recording spectrophotometer. The solid line shows the spectrum of the control enzyme, and the broken line shows the spectrum of the photooxidized enzyme.

615

DISCUSSION

Although ribulose bisphosphate carboxylase was obtained in a homogeneous state from higher plants more than 15 years ago, the relationship of structure to function is

06-

the other histidine residue is still available for modification by diethyl pyrocarbonate. To investigate this further, the photooxidized enzyme was first protected with competitive inhibitors like sedoheptulose 1.7-bisphosphate and 2-carboxy-D-mannito1 1,6-bisphosphate (22, 23). It has been shown that both of these inhibitors quantitatively protect the modification of one histidine residue out of two available for modification and since these are competitive inhibitors with respect to RuBP, the residue is presumably at or near the active site (13). If the pyridoxal phosphate-photooxidized residues were outside the domain of the active site, then there should be no protection by these competitive inhibitors against modification by diethyl pyrocarbonate. The results presented in Table V suggest that the residues modified by pyridoxal phosphate-dependent photooxidation are indeed at or near the active site since they are protected by either competitive inhibitor. The number of histidine residues modified by [3H]diethyl pyrocarbonate remains around 8 after various times of illumination. In the control experiment, these inhibitors protected about seven residues per 70,000-dalton re-

01

300

320 340 360 360 WAVELENGTH (nm)

FIG. 5. Absorption spectrum of photooxidized RuBP carboxylase with (---) and without (- - -) prior modification by diethyl pyrocarbonate (DEP). Enzyme (1 mg/ml) was modified with 1 mM DEP in Mops buffer, pH 6.2, containing 1% ethanol for 15 min. The control lacked only DEP. Samples were transferred to Bicine (pH 7.6) containing 10 mM MgClz, 10 mM HCO,, and 0.4 mM EDTA. Both control and DEPmodified enzyme were photooxidized for 30 min under conditions described under Materials and Methods at 1 mM pyridoxal phosphate except that DTT was not present. A control with pyridoxal phosphate was kept in the dark for the same time and showed no absorption at 325 nm (Fig. 4). All samples were dialyzed against 50 mM sodium phosphate (pH 7.6) to remove excess pyridoxal phosphate and the spectra run in a Cary 14 spectrophotometer.

616

BHAGWAT

AND

still poorly understood. Only one functional residue has been firmly placed in the primary structure of the spinach enzyme-Lyszol which binds COB during activation (25,26). In addition, LysiT5 is modified by two excellent affinity labels and preferentially by pyridoxal phosphate (27). Modifications of one or more essential arginines (28,29) and one essential tyrosine (30,31) and histidine (12,13) have also been described. In the present research we have attempted to localize the essential histidine in the spinach enzyme. In this approach pyridoxal phosphate was used at a concentration that would ensure preferential binding to lysine17s and the resultant Schiff base was subjected to photooxidation (4). Under these conditions, competitive inhibitors not only reduced the photoinactivation but protected one histidine/large subunit against subsequent modification by diethyl pyrocarbonate throughout the entire course of photooxidation. The available evidence suggests then that LyslT5 is in close proximity to the essential histidine and that both are at the active site. Using two other analogues of pyridoxal TABLE REACTION

OF [3H]D~~~~~~

IV PYROCARBONATE

([H3]DEP) WITH PYRIDOXAL PHOSPHATE PHOTOOXIDIZED ENZYME” Time of irradiation (min)

10 30 50

% of initial carboxylase activityb 100 65 22 10

mol [3H]DEP/mol of enzyme 16.44 12.13 10.1 9.09

a Photooxidation for the indicated time was done in Bicine, pH 7.6, containing 10 mM HCO; and 10 mM MgClz at an enzyme concentration of 640 pg/ml. The remaining carboxylase activity was measured as described under Materials and Methods. Modification with 0.5 mM [3H]diethyl pyrocarbonate was done at pH 7.0 for 10 min. Excess labeled reagent was removed by extensive dialysis against 67 mM phosphate buffer, pH 7.6. *Controls were incubated in the dark with 1 mM pyridoxal phosphate.

MC FADDEN TABLE

V

REACTION BETWEEN [3H]D~~~~~~ PYROCARBONATE AND PYRIDOXAL PHOSPHATE PHOTOOXIDIZED ENZYME IN THE PRESENCE OF COMPETITIVE INHIBITORS~

Time of irradiation (min)

mol [3H]DEP/mol of enzyme* % of initial activity

-

100 64 30 20 10

10 20 30 40

+SBP

+CMBP

10.1 9.8 8.7 8.6 8.6

10.8 9.6 8.3 8.3 8.5

“A control incubated without illumination and without protective agents in the presence or absence of 1 mM pyridoxal phosphate showed 17 mol of [3H]DEP bound/mol of enzyme. b The enzyme at 500 rg/ml was photooxidized at 1 mM pyridoxal phosphate for the indicated time. An aliquot was withdrawn for activity measurement. The remaining enzyme from each treatment was treated with either 2 mM sedoheptulose 1,7-bisphosphate (SBP) or 2 mM 2-carboxy-D-mannitol 1,6-bisphosphate (CMBP) for 15 min at 30°C. This was followed by [3H]DEP treatment at a concentration of 1 mM for 15 min at 30°C. Excess DEP was removed by extensive dialysis.

phosphate, the specificity of the reaction has also been shown. Neither pyridoxal nor pyridoxamine phosphate was nearly as effective, suggesting that both a phoshatebinding site and the e-amino group of LysiT5 are required for optimal binding and photooxidative capacity of pyridoxal phosphate. Although the present results establish that up to eight diethyl pyrocarbonate-reactive histidine residues/molecule are lost in concordance with activity during pyridoxal phosphate-dependent photooxidation, the exact mechanism is unknown. In related work the spectral change from 432 to 325 nm upon photooxidation, seen also in the present studies, has been attributed to addition of the nearby photooxidized histidyl

residue

to the )C=N-

of the

Schiff base (7, 10). Our results show that modification of the imidazolyl moiety of the essential histidine residue prior to ir-

ACTIVE

SITE

OF RIBULOSE

radiation in the presence of pyridoxal phosphate indeed eliminates the absorption maximum at 325 nm. In conclusion, the results presented establish that LysiT5 is near an active-site histidine. Recently we have suggested that a histidyl residue participates in the deprotonation of RuBP to yield a carbanion which may either react with COz or tetranitromethane (14). Also discussed was the participation of a proton donor (pK,: 8.6) in the carboxylative mechanism. This proton donor may be LysiT5; alternatively, this lysine residue may facilitate binding of RuBP or other anionic reaction intermediates. Whatever the interpretation, it will be of interest to determine whether the proximal histidyl and lysyl residues are close to one another in the peptide backbone or in the tertiary structure as a consequence of folding of the large subunit. ACKNOWLEDGMENT This research was supported in part by NIH Grant GM-19,972. We thank Dr. J. S. Nishimura for the generous gift of [3H]diethyl pyrocarbonate.

F. J., AND TOLBERT, N. E. (1977) Biophys. 179, 279-288. 2. WHITMAN, W., AND TABITA, F. R. (1976)Biochem. Biophys. Res. Cwmmun. 71, 1034-1039. 3. PAECH, C., RYAN, F. J., AND TOLBERT,

12. BHAGWAT,

Plant Physiol

57, 554.

13. SALUJA,

Arch.

6. DAVIS, L. C., BROX, L. W., GRACY, R. W., RIBEREAUGAYON, G., AND HORECKER, B. L. (1970)

Arch. B&hem.

Biophys.

140, 215-222.

7. GREENWELL, P., JEWETT, S. L., AND STARK, G. R. (1973) J. Biol Chem. 248, 5994-6001. 8. HUCHO, F., MARKAU, U., AND SUND, H. (1973) Eur.

J. Biochem. 32, 69-75. T., TORAYA,

J. B&hem.

T., AND FUKUI,

101.341-347.

S. (1979) Eur.

A.,

14. BHAGWAT,

S., AND

RAMAKRISHNA,

J. (1981)

Biophys. Acta 662,181-189. AND MCFADDEN,

chemistry

B. A.

(1982)

Birr

21, 89-95. A. S., AND MCFADDEN,

B. A.

(1983)

Arch. Biochem. Biophys. 223, 604-609. 15. BHAGWAT,

A.

S., AND MCFADDEN,

B. A.

(1982)

FEBS L&t. 145, 313-316. 16. BERHOW, M. A., SALUJA, A., AND MCFADDEN, B. A. (1982) Plant Sci. Z&t. 27, 51-57. 17. STOCK, A., ORTANDERI, F., AND PFLEIDERER, G. (1966) B&hem. Z. 344, 353-360. 18. MILES, E. W. (1977) in Methods in Enzymology

(Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 47, pp. 431-442, Academic Press, New York. 19. MCFADDEN,

20. 21. 22.

24. 25. 26.

253, 7866-7873. 5. RIPPA, M., AND PONTREMOLI, S. (1969) B&hem. Biophys. 103, 112-118.

A.

B&hem.

N. E. (1976)

4. PAECH, C., AND TOLBERT, N. E. (1978) J. Biol Chew.

9. NIHIRA,

463-468. A., AND MCFADDEN, B. A. (198O)Biochem. Biophys. Res. Commun 94,1091-1097.

11. SALUJA,

C. RYAN,

Arch. B&hem.

617

CARBOXYLASE

10. COZZANI, I., SANTONI, C., JORI, G., GENNARI, G., AND TAMBURRO, A. N. (1974) B&hem. J. 141,

23.

REFERENCES 1. PAECH,

BISPHOSPHATE

B. A.,

TABITA,

F. R., AND

KUEHN,

G. D. (1975) in Methods in Enzymology (Wood, W. A., ed.), Vol. 42, pp. 461-472, Academic Press, New York. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404427. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. SALUJA, A. K., AND MCFADDEN, B. A. (1978) FEBS Lett. 96, 361-363. ROACH, D., GOLLNICK, P. D., AND MCFADDEN, B. A. (1983) Arch. B&hem. Biophys. 222, 8794. SUGIYAMA, T., AND AKAZAWA, T. (1970) Biochemistry 9, 4499-4505. LORIMER, G. H. (1979) J. Biol. Chem. 254, 55995601. MIZIORKO, H. M. (1979) J. Biol. Chem 254, 270272.

27. SPELLMAN, M., TOLBERT, N. E., AND HARTMAN, F. C. (1979) Abstr. Papers, 178th National Meeting of the American Chemical Society, Washington, D. C., Sept. 1979, Biol. 3. 28. PUROHIT, K., MCFADDEN, B. A., AND LAWLIS, V. B. (1979) Arch. Microbial 121, 75-82. 29. LAWLIS, V. B., AND MCFADDEN, B. A. (1978) Biochem. Biophys. Res. Commun 80, 580-585. 30. ROBISON, P. D., AND TABITA, F. R. (1979) B&hem. Biophys. Res. Cmmun 88, 89-91. 31. BHAGWAT, A. S. (1982) Plant Sci. Lett. 27, 345353.