EFFICIENT EXPRESSION OF THE GENE FOR SPINACH PHOSPHORIBULOKINASE IN PICHIA-PASTORIS AND UTILIZATION OF THE RECOMBINANT ENZYME TO EXPLORE THE ROLE OF REGULATORY CYSTEINYL RESIDUES BY SITE-DIRECTED MUTAGENESIS

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regu lated in higher plants by thioredoxin-mediated thiol-disulfide exchang e in a light-dependent manner. Prior attempts to overexpress the highe r plant PRK gene in Escherichia coli for structure-function studies ha ve been hampered by sensitivity of the recombinant protein to proteoly sis as well as toxic effects of the protein on the host. To overcome t hese impediments, we have spliced the spinach PRK coding sequence imme diately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now ex pressed, in response to methanol, at 4-6% of total soluble protein, wi thout significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion -exchange and dye-affinity chromatography and is shown to be electroph oretically and kinetically indistinguishable from the authentic spinac h counterpart. Site-specific replacement of all of PRK's cysteinyl res idues (both individually and in combination) demonstrates a modest cat alytically facilitative role for Cys-55 (one of the regulatory residue s) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at po sition 55 display non-hyperbolic kinetics relative to the concentratio n of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and en hances kinase activity, presumably reflecting conformational differenc es between the position 55 mutants and wild-type enzyme. Catalytic com petence of the C16S-C55S double mutant proves that mere loss of free s ulfhydryl groups by oxidative regulation cannot account entirely for t he accompanying total inactivation.