| This thesis examines the minimal structural and functional requirements of the enzymic catalyst, protein disulfide isomerase (PDI). PDI is an essential protein in Saccharomyces cerevisiae, required for its ability to isomerize non-native disulfide bonds. Thus, proteins with PDI activity can be isolated by a powerful genetic selection. Eukaryotic protein disulfide isomerases are homologous to thioredoxin (Trx), a ubiquitous small (12 kDa) dithiol-disulfide oxidoreductase. Each PDI molecule contains two Trx domains per polypeptide chain and two other domains unrelated to Trx.;Wild-type Trx is unable to complement pdil$Delta$ S. cerevisiae. However, randomization of the CGPC active site to all twenty amino acids (i.e., CXXC Trx) yields enzymes--CWGC, CVWC, and CGHC--that can substitute for PDI in S. cerevisiae. The redox inactive CGPS Trx mutant also complements pdil$Delta$ S. cerevisiae. Each of the mutant CXXC enzymes has an elevated reduction potential compared to wild-type Trx ($Esp{circprime} = -0.270$ V). pH titrations of the CWGC and CVWC mutants monitored by $sp1$H- and $sp{13}$C-nuclear magnetic resonance (NMR) spectroscopy revealed that the first cysteine of the CXXC motif has a lowered p$Ksb{rm a}$ relative to wild-type Trx. These results confirm the hypothesis that the essential function of PDI resides in its isomerase activity, which is catalyzed by the dithiol form of the CXXC motif.;Mutants of Asp26 of Trx--D26N and D26L--have altered catalytic properties from the wild-type enzyme. Kinetics experiments with these mutant enzymes show that Asp26 is a general acid/base catalyst in the disulfide reduction and dithiol oxidation activities of Trx. Further, the mutation of Asp26 simplifies the pH-titration behavior of reduced Trx, revealing the presence of microscopic p$Ksb{rm a}$'s in the active-site of the reduced wild-type enzyme.;The experimental results presented in this Thesis illuminate the minimum functional group requirements for enzymic catalysis of disulfide bond isomerization. The interplay of the biophysical properties of the CXXC motif explains the wide presence in nature of CXXC--containing enzymes of different functions. |
