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Titolo:
Unexpected crucial role of residue 225 in serine proteases
Autore:
Guinto, ER; Caccia, S; Rose, T; Futterer, K; Waksman, G; Di Cera, E;
Indirizzi:
WashingtonAUniv, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 US Washington Univ St Louis MO USA 63110 Mol Biophys, St Louis, MO 63110 US
Titolo Testata:
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
fascicolo: 5, volume: 96, anno: 1999,
pagine: 1852 - 1857
SICI:
0027-8424(19990302)96:5<1852:UCROR2>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
NA+ BINDING-SITE; SUBSTRATE-SPECIFICITY; EVOLUTIONARY DIVERGENCE; CRYSTAL-STRUCTURE; THROMBIN; TRYPSIN; CHYMOTRYPSIN; CONSERVATION; SEQUENCE; ALPHA;
Keywords:
complement; molecular evolution; thrombin; water;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
34
Recensione:
Indirizzi per estratti:
Indirizzo: Di Cera, E WashingtonOUniv, Sch Med, Dept Biochem & Mol Biophys, Box 8231,St Louis, M Washington Univ Box 8231 St Louis MO USA 63110 231, St Louis, M
Citazione:
E.R. Guinto et al., "Unexpected crucial role of residue 225 in serine proteases", P NAS US, 96(5), 1999, pp. 1852-1857

Abstract

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparableor greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and aas driven by a significant gain (up to 50-fold) incatalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

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Documento generato il 01/04/20 alle ore 18:01:28