pH dependence of the peptide thiol-disulfide oxidase activity of six members of the human protein disulfide isomerase family.

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds, a process which in vivo is one of the rate limiting steps of protein folding and which is facilitated by the enzyme protein disulfide isomerase (PDI). Higher eukaryotes have multiple members of the PDI family, for example, seventeen human PDIs have been reported to date. With multiple members of the same family being present, even within the same cell, the question arises as to what differential functions are they performing? To date there has been no systematic evaluation of the enzymological properties of the different members of the PDI-family. To address the question of whether different PDI family members have differing thioldisulfide chemistry, we have recombinantly expressed and purified six members of the family, PDI, PDIp, ERp57, ERp72, P5, and PDIr from a single organism, human. An examination of the pH-dependence and nature of the rate limiting step for the peptide thiol-disulfide oxidase activity of these enzymes reveals that, with the exception of PDIr, they are all remarkably similar. In the light of this data potential differential functions for these enzymes are discussed.

A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases.

The pK(a) values of the CXXC active-site cysteine residues play a critical role in determining the physiological function of the thioredoxin superfamily. To act as an efficient thiol-disulphide oxidant the thiolate state of the N-terminal cysteine must be stabilised and the thiolate state of the C-terminal cysteine residue destabilised. While increasing the pK(a) value of the C-terminal cysteine residue promotes oxidation of substrates, it has an inhibitory effect on the reoxidation of the enzyme, which is promoted by the formation of a thiolate at this position. Since reoxidation is essential to complete the catalytic cycle, the differential requirement for a high and a low pK(a) value for the C-terminal cysteine residue for different steps in the reaction presents us with a paradox. Here, we report the identification of a conserved arginine residue, located in the loop between beta5 and alpha4 of the catalytic domains of the human protein disulphide isomerase (PDI) family, which is critical for the catalytic function of PDI, ERp57, ERp72 and P5, specifically for reoxidation. An examination of the published NMR structure for the a domain of PDI combined with molecular dynamic studies suggest that the side-chain of this arginine residue moves into and out of the active-site locale and that this has a very marked effect on the pK(a) value of the active-site cysteine residues. This intra-domain motion resolves the apparent dichotomy of the pK(a) requirements for the C-terminal active-site cysteine.

Defining the domain boundaries of the human protein disulfide isomerases.

The protein disulfide isomerase (PDI) family of folding catalysts are constructed from combinations of redoxactive and redox-inactive domains, all of which are probably based on the thioredoxin fold. To understand the function of each domain in the variety of catalytic reactions that each family member can perform (to differing extents), the domain boundaries of each family member must be known. By using a technique based on sequence alignments and the known structure of the a and b domains of human PDI, we generated a large number of domain constructs for all six redox-active human PDIs: PDI, PDIp, ERp72, ERp57, P5, and PDIr. The ability to generate significant amounts of soluble protein in E. coli from most of these domain constructs strongly indicates that the domain boundaries are correct. The implications for these domain boundaries on the tertiary structure of the human PDIs are discussed.