THE NATURE AND ALTERNATE FATES OF THE RIBULOSE 1,s- BISPHOSPHATE (RuBP) CARBOXYLASE/OXYGENASE (Rubisco) OXYGENATION INTERMEDXATE.

Mark R. Harpel, Yuh-Ru Chen, and Fred C. Hartman Protein Engineering Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

1. Introduction

Partitioning of carbon flow between photosynthesis or photorespiration reflects reactivity of Rubisco's RuBP-enediol intermediate with either C02, to form two molecules of 3- phospho-D-glycerate (PGA), or 0 2 , to form one molecule each of PGA and phosphoglycolate (PGyc). Although the enzyme's carboxylation reaction has been widely studied, the mechanism of oxygenation and the basis for CO;! versus 02 discrimination are still poorly understood; historically, confiiation of the putative oxygenation intermediate has proven elusive (see refs. 1-4 for reviews).

Functional and structural studies implicate active-site Lys329 and Glu48 of R. mbrum Rubisco in promoting addition of C02 to the RUBP-enediol (reviewed in refs. 1-2 & 5-6). In particular, substitution of either side chain by site-directed mutagenesis severely impairs carboxylation activity and selectivity for carboxylation versus oxygenation, but has lesser impact on the initial enediol-formation step in catalysis and on the ability to process purified carboxyketone carboxylation intermediate. Precise catalytic roles for these residues in oxygenation are unclear.

Two novel 02-dependent side products generated by the K329A and E48Q mutants provide insight into Rubisco's oxygenase intermediate and the roles of Lys329 and Glu48 in oxygenation (6-8). Formation of these side products and their further processing by wild-type, K329A, and E48Q Rubiscos show that Lys329 and Glu48 facilitate forward processing of the oxygenase intermediate to PGA and PGyc, despite the negative impact on photosynthetic efficiency.

2. Procedures

2.1 Materials Wild-type R. rubrum Rubisco was purified as previously described (9). The mutated R. d r u m rbc genes encoding E48Q and K329A Rubiscos were heterologously expressed in E. coli MV1190 and purified as previously described (10-11). [1-3H]RuBP was prepared from [2-3H]glucose (12). [ 1-3HlDiBP was isolated by anion-exchange chromatography from reactions of [l-X€jRuBP with E48Q in the presence of 120 mM sodium borate.

MASTER

-,

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or use- fulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any s p cific commercial product, process, or service by trade name, trademark, manufac-. turer, or otherwise does not necessarily constitute or imply its endorsement, recom- mendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

2.2 Product Analyses Radiolabeled reaction products were identified by anionexchange chromatography (13), in which deproteinated reaction mixtures were applied to a column of Mono Q (Pharmacia HR 5/5) and eluted with a gradient of NH4Cl at pH 8 in the presence of 10 mM sodium borate. Radioactivity was monitored by in-line scintillation counting. Specified reactions were depleted of oxygen using protocatechuate and protocatechuate 3,4-dioxygenase (13).

E e lo00

0

3. ResuIts and Discussion

CTBP i \I 60 min 180 min

XUBP - lo00 - 1

I 0 - . . . . I . , , - I . . .I. < I . I . I . . L . I . . . . I . - a . I . . , 7 2ooo

;ti,,

d.. . ' .e -

. C) E48Q + borate

3.1 02-Pependent Side Products Although inefficient at catalyzing RuBP carboxylation, E48Q and K329A transform RUBP to multiple aberrant products (Fig. 1). Two side products, xylulose- 1,5-bisphosphate (XuBP) and l-deoxy-~-gZycero-2,3-pentodidose-5-phosphate (DiMP), derive from misprocessing of the RuBP-enediol intermediate (1 1, 14). Formation of DiBP and CTBP requires 0 2 and generates H202 (7-8). These compounds therefore derive from the peroxyketone previously proposed (15) as Rubisco's oxygenase intermediate (Fig. 2).

XUBP

I.. . 'y t

Reaction time courses and chemical trapping of DiBP by borate (Fig. 1A-C for E48Q and Fig. 1D-F for K329A) indicate that DiBP is a transient in reactions catalyzed by each mutant. Whereas E48Q degrades DiBP to C-C scission products, K329A produces CTBP by a rearrangement mechanism (Fig. 2), as demonstrated by isotope-lab,eling studies (8).

. 30min I lo00 - DiBP

O d i ~ I . . . .

P

700- H-C-OH ~ H @ ~ 3 ~ + I

HzO + H+ *coo- CH~OPO~' PGA 2 PCyc

C H ~ O P O ~ ~ I

HO-*C-W-

€I-?-OH C'O I r"/ CH20POa2 C H Z O P O ~ ~

H+ CHaOPO3' peroxyketone Hoi bisphosphate I P H z O l HOfS-COO'

H-?-OH CH~OFO~'

(D-glycem-2,3-pcntose ~ k u b o x y t e ~ w

H - S O H I

CH~OPO~' DiBP CTBP

1J.~phoqh.*) 1Pbtspbmphate)

Figure 2 : Fates of Rubiso's peroxyketone oxygenase intermediate. The "upper" pathway represents normal oxygenation.

- 500 ; 400-D) + K329A I 4 o o L ~ ) Purified DBP I

I I t . 3 200 - 200 - ,I - mg

, . ( + o t - 0 2 ) I '

P

-

. n 5+ * _ _ _ _ _ _ _ _ - - - - - - - - - _ _ _ _ _ _ _ - - - - - - - - - - O - l - ~ ' ' - ~ ~ * ~ ~ ~ - 0 - * l ' . . r - l a * " . I ' *- 0

B) f No Enzyme 400 - E) + Wild-Type . (+0r-O2)

400-

E - & V 200 - 200 -

0 . . . . l . . . . I . . . . l . . , . I . . . ' 1 . ' . ' I ' . ' I . . ' . 0 & . 1 * * * , 1 - 1

. F) Authentic Mixture 400- CTBP

. C ) + W Q 400-

\ DiBP 200 - 200 -

00"' 'io ' ' 'io '"',"' ' '4 ' . 'io " 'Q6 I ' 70-m O

Time (mh) Figure 3: Turnover of isolated [ 1-3HIDiBP. (A) Purified starting material. (B) 24 h incubation of DiBP (2.5 pM) without enzyme. (C) E48Q reaction as in Fig. lA, except with 1 mM borate, 25 mM NH&l, and 5 pM DiBP (no RuBP) and quenched after 24 h. (D) K329A reaction as in Fig. lD, except with 0.5 rng/mL K329A, 20 mM NaHC03,7 mM borate, 130 mM NaCl, 2.5 pM DiBP (no RuBP) and quenched after 3 h. (E) reaction as in (D), except with 0.05 mg/mL wild-type. (I?) mixture of isolated DiBP and CTBP.

3.2 DiBP Turnover Direct assay of purified DiBP confirms its assignment as an alternate substrate for Rubisco variants. Distinct from spontaneous degradation (Fig. 3B), E48Q converts [1-3HJDiBP to [3H]PGyc (and presumably PGA) (Fig. 3B) and converts [SAHIDiBP to (3HJPGA (and

presumably PGyc) (not shown) by C-C cleavage. In contrast, CTBP is generated, independent of 0 2 , by K329A and wild-type Rubisco (Fig. 3D,E), although neither DiBP nor CTBP accumulates during wild-type m o v e r of RUBP. 3.3 summary 1) The novel Ordependent side products, DiBP and CTBP, provide direct evidence for the peroxyketone intermediate in Rubisco’s oxygenase pathway and serve as indicators of misprocessing . 2) Because the peroxyketone intermediate can suffer multiple fates (Fig. 2), it must be stabilized by Rubisco to mitigate formation of these side products. Physiologically, PGA and PGyc would be preferred products over DiBP and CTBP; whereas PGA can be used for carbon assimilation and some of the carbon lost to PGyc can be recovered through the glycolate pathway (although at a cost of energy; reviewed in refs. 4 & 16), DiBP and CTBP would likely be dead-end products for photosynthetic organisms. 3) Generation of DiBP and CTBP by K329A and E48Q supports roles for Lys329 and Glu48 in promoting oxygenation, as well as carboxylation, by properly activating the enediol and stabilizing subsequent intennediates. 4) Alternate pathways for DiBP processing indicate differential stabilization and activation of the dicarbonyl, once formed, to favor either rearrangement or C-C cleavage. 4. Acknowledgments %*

This research was sponsored by the USDOE Office of Health and Environmental Research under contract DE-ACO584OR21400 with Lockheed Martin Energy Systems, Inc.

1

References

1 Hartman, F. C. & Harpel, M. R (1993) Advances Enzymol. 67,l-75 2 Hartman, F. C. & Harpel, M. R (1994) Annu. Rev. Biochem. 63,197-234 3 Schloss, 5. V. (1990) in Enzymatic and Model Carboxylation and Reduction Reactions

for Carbon Dioxide Utilization (Aresta, M. & Schloss, 3. V., eds.) pp. 321-345, IUuwer Academic Publishers, The Netherlands

4 Andrews, T. J. & Lorimer, G. H. (1987) in The Biochemistry of Plants (Hatch, M. D. & Boardman, N. K., e&.) Vol. 10, pp. 131-218, Academic Press, New York

5 Schneider, G., Lindqvist, Y. & Brhden, C.-I. (1992) Annu. Rev. Biophys. Biomol.

6 Hartman, F. C., Harpel, M. R. & Chen, Y.-R., these proceedings 7 Chen, Y.-R & Hartman, F. C. (1995) J. Biol. Chem; 270,11741-11744 8 Harpel, M. R, Serpersu, E. H., Lamerdin, J. A, Huang, 2.-H., Gage, D. A. &

Hartman, F. C. (1995) Biochemistry 34, in press 9 Schloss, J. V., Phares, E. F., Long, M. V., Norton, I. L., Stringer, C. D. &

Hartman, F. C. (1982) Methods Enzymol. 90,522-528 10 Larimer, F. W., Mural, R J. & Soper, T. S. (1990) Protein Eng. 3,227-231 11 Harpel, M. R, & Hartman, F. C. (1994) Biochemistry 33,5553-5561 12 Kuehn, G. D., & Hsu, T.-C. (1978) Biochem. J. 175,909-912 13 Harpel, M. R., Lee, E. H. & Hartman, F. C. (1993) Anal. Biochem. 209,367-374 14 Lee, E. H., Harpel, M. R., Chen, Y.-R. & Hartman, F. C. (1993) J. Biol. Chem.

15 Lorimer, G. H., Andrews, T. J. & Tolbert, N. E. (1973) Biochemistry 12, 18-23 16 Ogren, W. L. (1984) Annu. Rev. Plant Physiol. 35,415-442

Struct. 21,119-143

268,26583-26591