"Spinach phosphoribulokinase : cloning, sequencing, and site-directed m" by Sylvia Milanez
 

Doctoral Dissertations

Date of Award

8-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Richard J. Mural

Committee Members

Frank Larimer, Fred Hartman, Elizabeth Howell

Abstract

Phosphoribulokinase (PRK) is a key light-regulated Calvin cycle enzyme which is deactivated by the formation of a disulfide bond between sulfhydryl groups. To be able to examine the enzyme's catalytic and regulatory mechanisms with the technique of site-directed mutagenesis, an expression/mutagenesis system was developed. A γgt11 library was constructed from spinach leaf poly A+ RNA. Clones containing the entire mature-length enzyme were identified using rabbit polyclonal antibody raised against spinach PRK The DNA sequence encoding mature-length PRK was determined: it encodes 351 amino acids with a calculated mass of 39,232 daltons and has a pI of 5.92. A PRK clone was modified so the mature-length protein would be expressed from the prk gene when inserted into an expression/mutagenesis vector. The resulting phagmid, pSM100, was introduced into Escherichia coli MV1190 and PRK indistinguishable in activity and mobility on SDS-polyacrylamide gels from the native enzyme was detected in crude extracts of these transformed cells. The initial issue addressed using site-directed mutagenesis was whether any of the four PRK cysteinyl residues, particularly those involved in light-regulation, have a role in catalysis. Thus, all four cysteinyl residues were replaced (individually) by either serine or alanine. The activities of the eight mutant kinases were determined in crude cell-free extracts. Extracts of position 16 and 55 mutants were purified partially and the Km values for the PRK substrates ATP and ribulose-5-phosphate (Ru5P) were measured. Results using both crude and partially purified extracts indicate that the sulfhydryl groups of Cys16, Cys244, and Cys250 are non-essential for structure or catalysis. PRK substituted at position 55 with either serine or alanine, however, had ten fold lower Kcat and 4 to 8-fold higher Km for Ru5P than wild-type enzyme. This indicates Cys55 has a facilitative role in catalysis and may be part of the sugar substrate binding domain of PRK The Ser16 and Ser55 mutant proteins were also examined for their reactivity with the sulfhydryl reagent N-ethylmaleimide. The refractiveness of the Cys16 mutant protein and sensitivity of the wild-type and Cys55 mutant proteins to inactivation by NEM indicates Cys16 was the modified residue. The hyper-reactivity of Cys16 to NEM and a number of other reagents suggests that it is ako the initial target for attack by the in vivo oxidant (oxidized thioredoxin) which causes oxidative deactivation of the enzyme.

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