Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin.
Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, Ki, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min(-1), five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.
Dee
Derek R
DR
Biophysics Interdepartmental Group, University of Guelph, Guelph, Ont., Canada N1G 2W1.
Filonowicz
Shaun
S
Horimoto
Yasumi
Y
Yada
Rickey Y
RY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D017931:DNA Primers; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010434:Pepsin A