1996
DOI: 10.1074/jbc.271.8.4461
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Abstract: Tissue-type plasminogen activator (t-PA) has evolved to optimize cleavage of plasminogen (Plg) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2 subsites of t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly selective cleavage of protein… Show more

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Cited by 41 publications
(43 citation statements)
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“…However, an environment similar to that of the longchain subfamily could be partially created with the participation of the substrate. We observe, in fact, for the short-chain enzymes a tendency to have a hydrophobic bulky residue in position P2 (Tong et al, 1993;Ingallinella et al, 1998), whereas longchain enzymes tend to have either glycine or short side-chain residues such as Ala (Yasutake & Powers, 1981;Chang, 1986;Coombs et al, 1996). A bulky residue in position P2 together with the aliphatic methylene groups of R155 (NS3) and of the L231 side-chain (Sindbis and Semliki viruses) could contribute to shelter the aspartic acid side-chain, thereby favouring the formation of a catalytic triad machinery that more closely resembles that observed in long-chain enzymes.…”
Section: The Catalytic Triad and Substrate-binding Regionmentioning
confidence: 90%
“…However, an environment similar to that of the longchain subfamily could be partially created with the participation of the substrate. We observe, in fact, for the short-chain enzymes a tendency to have a hydrophobic bulky residue in position P2 (Tong et al, 1993;Ingallinella et al, 1998), whereas longchain enzymes tend to have either glycine or short side-chain residues such as Ala (Yasutake & Powers, 1981;Chang, 1986;Coombs et al, 1996). A bulky residue in position P2 together with the aliphatic methylene groups of R155 (NS3) and of the L231 side-chain (Sindbis and Semliki viruses) could contribute to shelter the aspartic acid side-chain, thereby favouring the formation of a catalytic triad machinery that more closely resembles that observed in long-chain enzymes.…”
Section: The Catalytic Triad and Substrate-binding Regionmentioning
confidence: 90%
“…Furthermore, it is possible that NS3 is more active on polyprotein substrates than it is using a peptide substrate, thereby being less discriminative against suboptimal P1 residues. Differences in specific activity using either polyprotein or peptidic substrates have been reported for other proteases such as CMV protease (38) or tPa (39). Using in vitro translated substrates based on the NS5A/NS5B junction we have estimated the specific activity of added purified protease to be in the order of k cat /K m ϭ 200,000 M Ϫ1 s Ϫ1 .…”
Section: Discussionmentioning
confidence: 99%
“…the S and SЈ subsites function independently. For example, simple additive behavior is seen with substrate hydrolysis by trypsin (16), chymotrypsin (17), tissue-type plasminogen activator (16), and subtilisin (18). Nonadditive interactions, i.e.…”
Section: Methodsmentioning
confidence: 99%