Eva H. Lee

Complete primary structure of ribulosebisphosphate carboxylase/ oxygenase from Rhodospirillum rubrum

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.

Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum by hybridization of site-directed mutants

The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity. Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit. Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987). This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain). To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue. Coexpression inEscherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually. Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule. This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation. In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stablized by elevated ionic strengths or by glycerol.

Transfer RNA chromatography on reversed phase five: Effect of cadimum ion on a queuine-type tRNA*

A sensitive method is described that detects an alteration in the structure of tRNA that is caused by cadmium but not by magnesium or zinc ions. The chromatographic system, RPC-5, separates Drosophila tyrosyl-tRNA into two fractions. These two isoacceptors differ by a single position in the anticodon where either a guanosine or queuine resides. Cadmium ions apparently interact with the tRNA and prevent the chromatographic separation. This is the first instance where cadmium is shown to cause a selective change in nucleic acid structure. The RPC-5 system seems to be uniquely useful in detecting such a change.

Site-directed mutagenesis to determine essential residues of ribulose- bisphosphate carboxylase of Rhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.

Use of Trinitrobenzene Sulfonate to Determine the pKa Values of Two Active-Site Lysines of Ribulosebisphosphate Carboxylase/Oxygenase1

Ribulose-P2 carboxylase (E.C* 4.1.1.39), which catalyzes the carboxylation of ribulose-P2 to yield two molar equivalents of D-3-phosphoglycerate, provides the only significant route by which atmospheric CO2 is converted to carbohydrate and hence is absolutely essential to all higher life forms (see ref. 1 for a thorough review). The carboxylation reaction is suppressed by O2, which is both a competitive inhibitor (2) and a substrate of ribulose-P2 carboxylase (3). In the presence of O2, the enzyme catalyzes the conversion of ribulose-P2 to one molar equivalent each of phosphoglycolate and 3-phosphoglycerate. Although multiple substrate specificities among enzymes are not unusual, the bifunctionality of ribulose-P2 carboxylase is perhaps unprecedented in that the two reactions catalyzed are the initial steps in competing metabolic pathways — photosynthetic assimilation of CO2 and photorespiration, an energy wasteful process without a known function which results in the release of previously fixed CO2. Suppression of the carboxylase reaction by O2 clearly accounts for the long-recognized inhibition of photosynthesis by O2, i.e. the Warburg effect (4–8). Minimization of the Warburg effect by cultivating plants under controlled atmospheres of reduced O2 or elevated CO2 levels results in substantially enhanced growth rates and yields (9). Obviously, if the carboxylase/oxygenase ratio of ribulose-P2 carboxylase could be enhanced either by genetic engineering or by chemical treatment, agronomic benefits would ensue.