The C-terminal Sequence Encodes Function in Serine Proteases

Abstract

Serine proteases of the chymotrypsin family have maintained a common fold over an evolutionary span of more than one billion years. Notwithstanding modest changes in sequence, this class of enzymes has developed a wide variety of substrate specificities and important biological functions. Remarkably, the C-terminal portion of the sequence in the protease domain accounts fully for this functional diversity. This portion is often encoded by a single exon and contains most of the residues forming the contact surface in the active site for the P1-P3 residues of the substrate, as well as domains responsible for the modulation of catalytic activity. The evolution of serine proteases was therefore driven by optimization of contacts made with the unprimed subsites of the substrate and targeted a relatively short portion of the sequence toward the C-terminal end. The dominant role of the C-terminal sequence should facilitate the identification of function in newly discovered genes belonging to this class of enzymes.Serine proteases of the chymotrypsin family feature a gamut of important physiological functions, ranging from digestive and degradative processes to blood clotting, cellular and humoral immunity, fibrinolysis, fertilization, and embryonic development (1). How this variety of functions emerged during evolution from a highly conserved three-dimensional fold (2) is incompletely understood. Enzymes involved in digestive and degradative processes, such as trypsin and chymotrypsin, are found from bacteria to human and are composed of a protease domain containing all epitopes for ligand recognition (3, 4). On the other hand, enzymes involved in more specialized functions such as blood coagulation, humoral immunity, and fibrinolysis are present almost exclusively in vertebrates and carry additional modules that confer more stringent specificity and localize the proteolytic function in space (5-7). A large repertoire of functions can be created by linking the protease domain to specialized functional modules, and most likely this strategy enabled the evolution of highly selective and specialized enzymes from primitive digestive proteases (8).The mosaic organization of the primary structure of many serine proteases has fostered the notion that the protease domain alone would not bear reliable signatures of function. Previous sequence analyses and functional characterizations of serine proteases have relied heavily on the architecture of non-protease domains (6,8,9). Evolutionary trees for the hepatocyte growth factor (HGF), 1 HGF activator, kallikreins, mast cell protease, and complement lineages have been constructed in this manner (7, 10 -13). However, Doolittle and Feng (5) were able to construct a reasonable evolutionary tree for clotting and fibrinolytic proteases from analysis of the protease domain only. This domain is the only structural component present in all members of the serine protease family. We therefore posed the question as to whether this domain would suffice to gain a predictive understanding of th...

Conserved Ser residues, the Shutter Region, and Speciation in Serpin Evolution

“…Together, this new dendrogram and the codon data mutually reinforce each other and point toward a heavy focus on speciation during the course of serpin evolution. It should also be noted that superpositions of dendrograms for the S1 family of chymotrypsin-like proteases (14) upon those for the serpin superfamily do not match proteases with their respective inhibitors. Neither dendrogramatic analysis (14) nor analysis of evolutionary markers (6) points toward a significant correlation between speciation FIG.…”

“…Thus, the serpins are more similar to each other and are more parsimonious with regard to sequence changes. Phylogenetically, serpins group according to species and not function, unlike the proteases (14,15). The main driving force behind serpin evolution was therefore the emergence of new phyla.…”

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

“…Evidence has been presented that the evolutionary divergence of serine proteases can be explained fully in terms of the C-terminal sequence of the catalytic domain (51). This is 1SFQ (9).…”

The Impaired Viability of Prostate Cancer Cell Lines by the Recombinant Plant Kallikrein Inhibitor

“…Protease Inhibition Assays-The inhibition of trypsin was used to evaluate the activity of the expressed protein as a model for enzymes with the same mechanism of action, called trypsintype enzymes, such as the kallikrein family (32)(33)(34). The assays were performed in a 96-well plate in a final volume of 250 l as described by Oliva et al (35).…”