Effect of amino acid substitution at the P(3) and P(4) subsites of fusion proteins on kex2 protease activity.
The effect of amino acid substitution at the P(3) and P(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction designed to allow cleavage with Kex2-660. Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P(3)-P(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys. As for Asp and Glu at P(4), they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the introduction of Asp at P(4) of the peptide substrate resulted in a large increase in K(m) and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P(4)-P(3). The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution. Thus Kex2 exhibited extended substrate specificity beyond P(2)-P(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.
Suntory Institute for Medicinal Research and Development, 2716-1 Akaiwa, Chiyoda-machi, Ohra-Gun, Gunma 370-0503, Japan. Yuji.Suzuki@suntory.co.jp
Y
Y
Suzuki
N
N
Ikeda
E
E
Kataoka
K
K
Ohsuye
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029742:Caenorhabditis elegans Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D043484:Proprotein Convertases; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; C060620:Glp-1 protein, C elegans; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae