The cogulation proteases interacts with their substrates in a complex manner.Their accion must be precisely regulated, otherweise will harm the organism.How rise their regulation and specificity?
Starting with Hydrolysis of a polypeptide chain,it requires proper recognition, orientation and binding of the substrate polypeptide backbone.Residues adjacent to the scissile bond have a significant impact on the rate of hydrolysis.
The nomenclature suggested by Schechter and Berger:Substrate amino acids are termed P (for peptide) and
the residues of the protease that interact with them are called S (for subsite). Substrate residues extending toward theN-terminus
of the scissile bond of substrate are numbered P2, P3, P4 and so forth. Conversely, substrate residues extending
toward the C-terminus are labeled P2', P3', P4' and onwards.For example, the P1 residue is bound in the S1 pocket.
Active site specificity, as described by the S and P nomenclature, fails to fully describe the physiologically relevant specificity of many S1 peptidases.Proteases of vertebrate blood coagulation utilize additional protein-protein and protein-lipid interactions to form ternary protein complexes.
The active forms of coagulation factors VIIa, IXa, Xaand activated protein C are examples of enzymes that employ extensive protein-protein interactions within the protease domain itself, as well as interactions between auxiliary domains.In contrast,thrombin is devoid of auxiliary domains, but exhibits a high degree of selectivity toward peptide substrates and similarly utilizes protein-protein interactions
So we can say that substrate specificity arises from contributions of subsites surrounding the active site, exosite interactions occurring within the protease domain and further interactions mediated by associated protein domains.
We find less than 20 a.a that are related to enzyme-substrate interactions. Some examples include residues 214-216, which are involved in formation of the antiparallel b-strand interaction between enzyme and substrate, and three disulfide bonds between residues 42 and 58, 168 and 182, and 191 and 220 that stabilize the entire domain. In both instances, these residues are highly conserved throughout the serine protease family and do not significantly modulate specificity.
P98139|192-431|FVII_rabbit PEVTGNMFCAGYLDGSK---DACKGDSGGPHATS--YHGTWYLTGVVSWG 213
Q9GLP2|214-448|PC_pig --ISENMLCAGILGDSR---DACEGDSGGPMVAS--FRGTWFLVGLVSWG 208
P00740|227-459|FIX_Homo --IYNNMFCAGFHEGGR---DSCQGDSGGPHVTE--VEGTSFLTGIISWG 206
P00743|234-466|FX_Bovis --ITPNMFCAGYDTQPE---DACQGDSGGPHVTR--FKDTYFVTGIVSWG 206
P00735|367-621|Thrombin_Bos --ITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVSWG 228
sp|P00767|CTRB_BOVIN --VTDVMICAG--ASGV---SSCMGDSGGPLVCQ--KNGAWTLAGIVSWG 216
sp|P00761|TRYP_PIG --ITGNMICVGFLEGGK---DSCQGDSGGPVVC----NG--QLQGIVSWG 202
: *:*.* .:* ****** . .. *::***
S1 subsite specificity
All coagulation proteases have trypsin-like specificity at the primary P1 position and prefer to hydrolyze peptide bonds on the C-terminal side of Arg residues. The mechanism by which specificity is generated in the S1 pocket.The P1-S1 nteraction is ionic in nature and involves formation of saltbridges between the guanidinium group of the Arg of substrate and the carboxylate group of Asp-189 in the S1 site.
P98139|192-431|FVII_rabbit PEVTGNMFCAGYLDGSK---DACKGDSGGPHATS--YHGTWYLTGVVSWG 213
Q9GLP2|214-448|PC_pig --ISENMLCAGILGDSR---DACEGDSGGPMVAS--FRGTWFLVGLVSWG 208
P00740|227-459|FIX_Homo --IYNNMFCAGFHEGGR---DSCQGDSGGPHVTE--VEGTSFLTGIISWG 206
P00743|234-466|FX_Bovis --ITPNMFCAGYDTQPE---DACQGDSGGPHVTR--FKDTYFVTGIVSWG 206
P00735|367-621|Thrombin_Bos --ITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVSWG 228
sp|P00767|CTRB_BOVIN --VTDVMICAG--ASGV---SSCMGDSGGPLVCQ--KNGAWTLAGIVSWG 216
sp|P00761|TRYP_PIG --ITGNMICVGFLEGGK---DSCQGDSGGPVVC----NG--QLQGIVSWG 202
: *:*.* .:* ****** . .. *::*** On the other hand, selectivity of the S2-S4-binding pockets varies widely within the coagulation proteases.
S2 subsite specificity
Selectivity at the S2 subsite is the most apparent feature in coagulation proteases. Discrimination of P2 side chains is mediated by loop insertions at positions 60 and 99. Relative to chymotrypsin, coagulation proteases possess larger loops at these two key positions.In FXa, the 99-loop presents a bulky tyrosine residue, Tyr-99, which occludes the S2 pocket and generates a preference for Gly side chains.In thrombin, an insertion of 10 amino acid residues at position 60 generates a preference for Pro at P2 that is sandwiched between two planar hydrophobic residues.
P98139|192-431|FVII_rabbit HWVVSAAHCFDKLSSLRNLTI-----VLGEHDLSEHE-GDEQVRHVAQLI 77
Q9GLP2|214-448|PC_pig SWVLTAAHCLDDY---KKLTV-----RLGEYDLRRRE-KWEVDLDIKEFL 75
P00740|227-459|FIX_Homo KWIVTAAHCVETG---VKITV-----VAGEHNIEETE-HTEQKRNVIRII 74
P00743|234-466|FX_Bovis FYVLTAAHCLHQA---KRFTV-----RVGDRNTEQEE-GNEMAHEVEMTV 75
P00735|367-621|Thrombin_Bos RWVLTAAHCLLYPPWDKNFTVDDLLVRIGKHSRTRYERKVEKISMLDKIY 85
sp|P00767|CTRB_BOVIN DWVVTAAHCGVTT-----SDV----VVAGEFDQGLET-EDTQVLKIGKVF 89
sp|P00761|TRYP_PIG QWVVSAAHCYKSR-----IQV----RLG-EHNIDVLE-GNEQFINAAKII 79
::::**** : . .
P98139|192-431|FVII_rabbit MPDKY--VPGKTDHDIALLRLLQPAALTNNVVPLCLPERNFSESTLATI- 124
Q9GLP2|214-448|PC_pig VHPNY--TRSTSDNDIALLRLAEPATFSQTIVPICLPDSGLSERELTRVG 123
P00740|227-459|FIX_Homo PHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTN-IFLKF- 122
P00743|234-466|FX_Bovis KHSRF--VKETYDFDIAVLRLKTPIRFRRNVAPACLPEKDWAEATLMTQ- 122
P00735|367-621|Thrombin_Bos IHPRYN-WKENLDRDIALLKLKRPIELSDYIHPVCLPDKQTAAKLLHAG- 133
sp|P00767|CTRB_BOVIN KNPKFS--ILTVRNDITLLKLATPAQFSETVSAVCLPSADEDFPAGMLC- 136
sp|P00761|TRYP_PIG THPNFN--GNTLDNDIMLIKLSSPATLNSRVATVSLPRS--CAAAGTEC- 124
.: . ** ::.* * : : . .:. S3 and S4 interactions
Binding of the P3 residue is generated by residues 192 and 216. Throughout the entire family of S1 peptidases, residue 216 is highly conserved as a Gly.Selectivity for P3 residues is poor in nearly all S1 peptidases as the enzyme-substrate interaction is limited due to solvent exposure of the P3 side chain.
Few S1 peptidases possess selectivity for P4 residues. The 172-loop and the side chain of 215 create the S4-binding pocket. The latter is highly conserved and generates a hydrophobic S4 pocket. Mutation of Trp-215 in thrombin has profound consequences on activity, specificity and structure.
P98139|192-431|FVII_rabbit RFSRVSGWG-QLLYRGA-----LARELMAIDVPRLMTQDCVEQSEHKPGS 168
Q9GLP2|214-448|PC_pig QETVVTGWGYRSEAKTN-----RSFILNFIKVPVAPHNECVQAMHNK--- 165
P00740|227-459|FIX_Homo GSGYVSGWG-RVFHKGR-----SALVLQYLRVPLVDRATCLRSTKFT--- 163
P00743|234-466|FX_Bovis KTGIVSGFG-RTHEKGR-----LSSTLKMLEVPYVDRSTCKLSSSFT--- 163
P00735|367-621|Thrombin_Bos FKGRVTGWGNRRETWTTSVAEVQPSVLQVVNLPLVERPVCKASTRIR--- 180
sp|P00767|CTRB_BOVIN ---ATTGWGKTKYNALK-----TPDKLQQATLPIVSNTDCRKYWGSR--- 175
sp|P00761|TRYP_PIG ---LISGWGNTKSSGSS-----YPSLLQCLKAPVLSDSSCKSSYPGQ--- 163
:*:* . * * *
P98139|192-431|FVII_rabbit PEVTGNMFCAGYLDGSK---DACKGDSGGPHATS--YHGTWYLTGVVSWG 213
Q9GLP2|214-448|PC_pig --ISENMLCAGILGDSR---DACEGDSGGPMVAS--FRGTWFLVGLVSWG 208
P00740|227-459|FIX_Homo --IYNNMFCAGFHEGGR---DSCQGDSGGPHVTE--VEGTSFLTGIISWG 206
P00743|234-466|FX_Bovis --ITPNMFCAGYDTQPE---DACQGDSGGPHVTR--FKDTYFVTGIVSWG 206
P00735|367-621|Thrombin_Bos --ITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVSWG 228
sp|P00767|CTRB_BOVIN --VTDVMICAG--ASGV---SSCMGDSGGPLVCQ--KNGAWTLAGIVSWG 216
sp|P00761|TRYP_PIG --ITGNMICVGFLEGGK---DSCQGDSGGPVVC----NG--QLQGIVSWG 202
: *:*.* .:* ****** . .. *::*** Exosite interactions
Two regions on the protease domain are involved in protein-protein interactions and are known to influence the properties of the active site. These regions are two exosites, I and II, located on opposing sides of the active site. Exosite I is composed of two stretches of amino acids at positions 30-40 and 70-80 in the first six-stranded b-barrel of the protease domain to the -east- of the active site.
P98139|192-431|FVII_rabbit ---------------IVGGKVCPKGECPWQAALMNG--STLLCGGSLLDT 33
Q9GLP2|214-448|PC_pig ---------------LVNGKQSPWGESPWQVILLDSK-KKLACGAVLIHV 34
P00740|227-459|FIX_Homo ---------------VVGGEDAKPGQFPWQVVLNG-K-VDAFCGGSIVNE 33
P00743|234-466|FX_Bovis ---------------IVGGRDCAEGECPWQALLVNEE-NEGFCGGTILNE 34
P00735|367-621|Thrombin_Bos ---------------IVEGQDAEVGLSPWQVMLFRKSPQELLCGASLISD 35
sp|P00767|CTRB_BOVIN CGVPAIQPVLSGLARIVNGEDAVPGSWPWQVSLQDST-GFHFCGGSLISE 49
sp|P00761|TRYP_PIG --FPTDD-----DDKIVGGYTCAANSIPYQVSLNSGS---HFCGGSLINS 40
:* * . . *:*. * **. ::
P98139|192-431|FVII_rabbit HWVVSAAHCFDKLSSLRNLTI-----VLGEHDLSEHE-GDEQVRHVAQLI 77
Q9GLP2|214-448|PC_pig SWVLTAAHCLDDY---KKLTV-----RLGEYDLRRRE-KWEVDLDIKEFL 75
P00740|227-459|FIX_Homo KWIVTAAHCVETG---VKITV-----VAGEHNIEETE-HTEQKRNVIRII 74
P00743|234-466|FX_Bovis FYVLTAAHCLHQA---KRFTV-----RVGDRNTEQEE-GNEMAHEVEMTV 75
P00735|367-621|Thrombin_Bos RWVLTAAHCLLYPPWDKNFTVDDLLVRIGKHSRTRYERKVEKISMLDKIY 85
sp|P00767|CTRB_BOVIN DWVVTAAHCGVTT-----SDV----VVAGEFDQGLET-EDTQVLKIGKVF 89
sp|P00761|TRYP_PIG QWVVSAAHCYKSR-----IQV----RLG-EHNIDVLE-GNEQFINAAKII 79
::::**** : . . In contrast, exosite II is located 10 A to the -west- of Asp-102 in the catalytic triad and is formed by a cluster of residues(93 165 230 233 236) on and about the C-terminal helix. Comparison of exosite residues in related coagulation proteases shows substantial variation in sequence.
Regulating specificity: the Na+-dependent allostery
Thrombin and other vitamin K-dependent clotting proteases are enzymes activated by monovalent cations and require Na+ for optimal catalytic activity . This property is also shared by proteases involved in immune response, but not by digestive and degradative enzymes. The Na+-binding site of thrombin is strategically located in close proximity to the primary specificity pocket, nestled between the 220- and 186-loops that contribute to substrate specificity in serine proteases. The Na+ binding sites of FXa and a PC are similarly located.
P98139|192-431|FVII_rabbit PEVTGNMFCAGYLDGSK---DACKGDSGGPHATS--YHGTWYLTGVVSWG 213
Q9GLP2|214-448|PC_pig --ISENMLCAGILGDSR---DACEGDSGGPMVAS--FRGTWFLVGLVSWG 208
P00740|227-459|FIX_Homo --IYNNMFCAGFHEGGR---DSCQGDSGGPHVTE--VEGTSFLTGIISWG 206
P00743|234-466|FX_Bovis --ITPNMFCAGYDTQPE---DACQGDSGGPHVTR--FKDTYFVTGIVSWG 206
P00735|367-621|Thrombin_Bos --ITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVSWG 228
sp|P00767|CTRB_BOVIN --VTDVMICAG--ASGV---SSCMGDSGGPLVCQ--KNGAWTLAGIVSWG 216
sp|P00761|TRYP_PIG --ITGNMICVGFLEGGK---DSCQGDSGGPVVC----NG--QLQGIVSWG 202
: *:*.* .:* ****** . .. *::***
P98139|192-431|FVII_rabbit EGCAAVGHVGVYTRVSRYTEWLSRLMR-- 240
Q9GLP2|214-448|PC_pig EGCGRLHNYGVYTKVSRYLDWIHGHIR-- 235
P00740|227-459|FIX_Homo EECAMKGKYGIYTKVSRYVNWIKEKTK-- 233
P00743|234-466|FX_Bovis EGCARKGKFGVYTKVSNFLKWIDKIMK-- 233
P00735|367-621|Thrombin_Bos EGCDRDGKYGFYTHVFRLKKWIQKVID-- 255
sp|P00767|CTRB_BOVIN SSTCSTSTPAVYARVTALMPWVQETLAAN 245
sp|P00761|TRYP_PIG YGCAQKNKPGVYTKVCNYVNWIQQTIAAN 231
..*::* *: