Oxidation of kinetically trapped thiols by protein disulfide isomerase.
The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1). PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Walker
K W
KW
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Gilbert
H F
HF
D000478:Alkylation; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D007535:Isomerases; D019704:Protein Disulfide-Isomerases