Crosslinking agents as active-site probes for phosphoribulokinase (PRK)

Journal Article · Wed May 01 00:00:00 EDT 1991 · Plant Physiology, Supplement; (United States)

OSTI ID:7160144

Brandes, H M; Hartman, F C ^[1]

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1. Oak Ridge National Lab., TN (United States) Univ. of Tennessee, Oak Ridge (United States)

The light-dependent, thioredoxin-mediated activation of plant PRK, discovered by B.B. Buchanan and colleagues, entails reduction of an intra-subunit disulfide at the active site composed of Cys16 and Cys55. Hyper-reactivity of the cysteinyl-16 sulfhydryl has been demonstrated by its preferential derivatization by chemically diverse reagents, e.g. iodoacetate, bromoacetylethanolamine phosphate, and methylnitrobenzenesulfonate. The authors are attempting to probe further the topology of the active site by exploiting the reactivity of Cys16. Covalent crosslinking agents should target Cys16, thereby introducing a moiety capable of labeling another proximal amino acid side chain. In the absence of exogenous thiols, difluorodinitrobenzene (FNB), difluoronitrophenylsulfone (FNPS), and diisothiocyanodisulfonate stilbene (DIDS) inactive PRK. Inactivation by FNPS or DIDS is partially reversed by DTT. Saturating levels of ATP provide protection from inactivation by each of the three reagents. In contrast, Ru5P accelerates inactivation of the enzyme by FNB and DIDS. These differential effects of the two substrates are consistent with physically distinct binding domains within the active site. Denaturing gel electrophoresis eliminates the possibility of intermolecular or intersubunit crosslinking. Cys 16 is implicated in the derivatization by the resistance of the Cys16-Ser site-directed mutant protein to each reagent.

• DOE Contract Number: AC05-84OR21400
• OSTI ID: 7160144
• Journal Information: Plant Physiology, Supplement; (United States), Vol. 96:1; ISSN 0079-2241
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PHOSPHOTRANSFERASES
BIOCHEMICAL REACTION KINETICS
SUBSTRATES
AROMATICS
ATP
CROSS-LINKING
CYSTEINE
FLUORINE COMPOUNDS
INACTIVATION
NITRO COMPOUNDS
REAGENTS
THIOLS
AMINO ACIDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ENZYMES
HALOGEN COMPOUNDS
KINETICS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERIZATION
PROTEINS
REACTION KINETICS
TRANSFERASES
550200* - Biochemistry