1. Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity
Gregg B Fields, Michal Tokmina-Roszyk, Manishabrata Bhowmick, Dorota Tokmina-Roszyk Anal Biochem . 2014 May 15;453:61-9. doi: 10.1016/j.ab.2014.02.024.
Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] had a melting temperature (Tm) of 36.2°C and was hydrolyzed efficiently by bacterial collagenase (k(cat)/K(M)=25,000s(-1)M(-1)) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.
2. Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2
Haruyuki Iefuji, Takuya Hirano, Kazuo Masaki, Shengbin Rao, Osamu Mizutani J Biosci Bioeng . 2011 Nov;112(5):441-6. doi: 10.1016/j.jbiosc.2011.07.013.
An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂ and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.
3. Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3)
H Nagase, C G Fields, G B Fields J Biol Chem . 1994 Aug 19;269(33):20952-7.
Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
