Unexpected crucial role of residue 225 in serine proteases

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 comparable or 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 inf luences 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 was driven by a significant gain (up to 50-fold) in catalytic 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.

A Structural Perspective on Enzymes Activated by Monovalent Cations

“…Remarkably, the conformation of this sequence relative to the bound K ϩ in the channel is very similar to the GYG sequence (residues 325-327) near the K ϩ binding site of pyruvate dehydrogenase kinase (14), the GFG sequence (residues 337-339) near the K ϩ binding site of branched chain ␣-ketoacid dehydrogenase kinase (13), and the KYG sequence (residues 224 -226) near the Na ϩ binding site of thrombin (42). Furthermore, mutation of Tyr in this sequence has very similar functional consequences in the K ϩ channel (50) and thrombin (51). This unexpected connection is a testimony to the basic similarity in the mechanism of M ϩ recognition that evolution has bestowed on proteins with widely different functions.…”

Murine serine proteases MASP-1 and MASP-3, components of the lectin pathway activation complex of complement, are encoded by a single structural gene

“…The catalytic triad composed of histidine, aspartic acid, and serine (Ser 195) is conserved. In addition, a serine (Ser 214) relevant to substrate binding 25 is conserved in guinea pig, mouse, rat, and rabbit, and a tyrosine (Tyr 225) typically found in Na + -activated allosteric enzymes 26 is found in rat, mouse, and cow C-terminal sequences. All potential N-glycosylation sites (characterised by the following sequence of four amino acids: Asn X(no Pro), Thr/Ser/Cys, no Pro) present in the human MASP-3 protein sequence are conserved in the overlapping sequences of all species studied (see Figure 5).…”

Crystal Structure of the Anticoagulant Slow Form of Thrombin

“…At the resolution limits of the crystal, the Na ϩ environment shows a conspicuous paucity of solvent molecules ( Fig. 2A), especially in the channel embedding the S1 specificity site that is highly hydrated in the fast form (20). Well defined density however exists for the backbone around the loop segments 220 -224 and 186 -187 that define the Na ϩ binding site (21).…”