key: cord-018460-wbtaoo0o
authors: Schomburg, Dietmar; Schomburg, Ida
title: SARS coronavirus main proteinase 3.4.22.69
date: 2013
journal: Class 3.4-6 Hydrolases, Lyases, Isomerases, Ligases
DOI: 10.1007/978-3-642-36260-6_3
sha: 
doc_id: 18460
cord_uid: wbtaoo0o

EC number 3.4.22.69 Recommended name SARS coronavirus main proteinase Synonyms 3C-like protease <2,3> [9,16,38,49,51] 3CL protease <2> [14,48] 3cLpro <1,2,3> [7,11,13,16,19,28,38,49,51] C30.004 (Merops-ID) Mpro SARS 3C-like protease <2> [17] SARS 3C-like proteinase <2> [15,18,27] SARS 3CL protease <2> [31] SARS 3CLpro <2> [49] SARS CoV main proteinase <2> [1,2,4,5] SARS CoVMpro <2> [33] SARS Mpro <2> [25] SARS coronavirus 3C-like protease <2> [48] SARS coronavirus 3C-like proteinase <2> [50] SARS coronavirus 3CL protease <2> [20] SARS coronavirus main peptidase <2> [23] SARS coronavirus main protease <2> [25] SARS coronavirus main proteinase <2> [5,33] SARS main protease <2> [12,25] SARS-3CL protease <2> [48] SARS-3CLpro <2> [29,50] SARS-CoV 3C-like peptidaseSARS-CoV 3C-like peptidase<2> [24] SARS-CoV 3C-like protease<1> [19] SARS-CoV 3CL protease <2> [22,30,44,46] SARS-CoV 3CLpro <2> [32,36,38,44,45] SARS-CoV 3CLpro enzyme <2> [11] SARS-CoV Mpro <2> [21,40] SARS-CoV main protease <2> [21,26,43] SARS-coronavirus 3CL protease <2> [8] SARS-coronavirus main protease <2> [47] TGEV Mpro coronavirus 3C-like protease <1> [19] porcine transmissible gastroenteritis virus Mpro severe acute respiratory syndrome coronavirus 3C-like protease <2> [41,42] severe acute respiratory syndrome coronavirus main protease <2> [21] severe acute respiratory syndrome coronavirus main proteinase <2> [33] CAS registry number 218925-73-6 37353-41-6

porcine transmissible gastroenteritis virus Mpro severe acute respiratory syndrome coronavirus 3C-like protease <2> [41, 42] severe acute respiratory syndrome coronavirus main protease <2> [21] severe acute respiratory syndrome coronavirus main proteinase <2> [33] CAS registry number 218925-73-6 37353- 2 Source Organism <1> alphacoronavirus [19] <2> SARS coronavirus [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50] <3> SARS coronavirus Tor2 (UNIPROT accession number: P0C6U8) [51] 3 Reaction and Specificity Catalyzed reaction TSAVLQ-/-SGFRK-NH 2 and SGVTFQ-/-GKFKK the two peptides corresponding to the two self-cleavage sites of the SARS 3C-like proteinase are the two most reactive peptide substrates. The enzyme exhibits a strong preference for substrates containing Gln at P1 position and Leu at P2 position.

Natural substrates and products S coronavirus polyprotein + H 2 O <2> (<2> 3CLpro processes the translated polyproteins to functional viral proteins [28] ) (Reversibility: ?) [28] P ? S Additional information <2> (<2> SARS-CoV 3CLpro mediates extensive proteolytic processing of two overlapping replicase polyproteins, pp1a (486000 Da) and pp1ab (790000 Da), to yield the corresponding functional polypeptides that are essential for SARSCoV replication and transcription processes [42] ; <2> the genomic RNA produces two large proteins with overlapping sequences, polyproteins 1a and 1ab, which are autocatalytically cleaved by two or three viral proteases to yield functional polypeptides. The key enzyme in this processing is SARS 3CL protease [31] ; <2> 3CLpro cleaves the replicase polyproteins at 11 sites with the conserved Gln-(Ser, Ala, Gly) sequences [49] ) (Reversibility: ?) [31, 42, 49 ] P ?

Substrates and products S (Ala-Arg-Leu-Gln-NH) 2 [15] P AAVLQ + SGF-NH 2 S ATVRLQAGNAT + H 2 O <2> (Reversibility: ?) [15, 27] P ATVRLQ + AGNAT S AVLQS-NH 2 + H 2 O <2> (Reversibility: ?) [15] P AVLQ + l-serinamide S AVLQSE-NH 2 + H 2 O <2> (Reversibility: ?) [15] P AVLQ + Ser-Glu-NH 2 S AVLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P AVLQ + SGF-NH 2 S DABCYL-Lys-Asn-Ser-Thr-Leu-Gln-Ser-Gly-Leu-Arg-Lys-Glu-EDANS + H 2 [15, 27] P FYPKLQ + ASQAW S GPFVDRQTAQAAGTDT-NH 2 + H 2 O <2> (<2> 1% of the activity with TSAVLQSGFRK-NH 2 [31] ) (Reversibility: ?) [31] P ? S KVATVQSKMSD + H 2 O <2> (Reversibility: ?) [15] P KVATVQ+ SKMSD S KVATVQSKMSD + H 2 O <2> (<2> weak activity [27] ) (Reversibility: ?) [27] P KVATVQ + SKMSD S KVATVQSKMSD-NH 2 <2> (<2> undecapeptide containing the non-canonical P3/P4 cleavage site of 3CL protease, 6% of the activity with TSAVLQSGFRK-NH 2 [31] ) (Reversibility: ?) [31] P ? S l-Thr-l-Ser-l-Ala-l-Val-l-Leu-l-Gln-4-nitroanilide + H 2 O <2> (Reversibility: ?) [48] P l-Thr-l-Ser-l-Ala-l-Val-l-Leu-l-Gln + 4-nitroaniline S LAVLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P LAVLQ + SGF-NH 2 S LQSG-NH 2 + H 2 O <2> (Reversibility: ?) [15] P Leu-Gln + Ser-Gly-NH 2 S MCAAVLQSGFR-Lys(Dnp)-Lys-NH 2 + H 2 O <2> (Reversibility: ?) [40] P MCAAVLQ + Ser-Gly-Phe-Arg-Lys(Dnp)-Lys-NH 2 S NRATLQAIASE + H 2 O <2> (<2> weak activity [27] ) (Reversibility: ?) [15, 27] P NRATLQ + AIASE S NVATLQAENVT + H 2 O <2> (<2> weak activity [27] ) (Reversibility: ?) [15, 27] P NVATLQ + AENVT S PATVLQAVGAC + H 2 O <2> (Reversibility: ?) [15] P PATVLQ + AVGAC S PHTVLQAVGAC + H 2 O <2> (Reversibility: ?) [27] P PHTVLQ + AVGAC S REPLMQSADAS + H 2 O <2> (<2> weak activity [27] ) (Reversibility: ?) [15, 27] P REPLMQ + SADAS S SAALQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P SAALQ + SGF-NH 2 S SAKLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P SAKLQ + SGF-NH 2 S SALLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P SALLQ + SGF-NH 2 S SATLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [15] P SATLQ + SGF-NH 2 S SAVAQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [ P Ser-Ala-Val-Leu-Gln + Leu-Gly-Phe-Arg-Lys S Ser-Ala-Val-Leu-Gln-Met-Gly-Phe-Arg-Lys + H 2 O <2> (Reversibility: ?)

[49] P Ser-Ala-Val-Leu-Gln + Met-Gly-Phe-Arg-Lys S Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys + H 2 O <2> (Reversibility: ?)

[49] P Ser-Ala-Val-Leu-Gln + Ser-Gly-Phe-Arg-Lys S TAVLQSGF-NH 2 + H 2 O <2> (Reversibility: ?) [ [8] P o-aminobenzoyl-TSAVLQ + SGFRY(3-NO 2 )G S Additional information <2> (<2> a 3CLpro mechanism utilizes an electrostatic trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base catalyzed deacylation reaction [7] ; <2> complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160 000 possible naturally occurring tetrapeptides. P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln [6] ; <2> SARS-CoV 3CLpro mediates extensive proteolytic processing of two overlapping replicase polyproteins, pp1a (486000 Da) and pp1ab (790000 Da), to yield the corresponding functional polypeptides that are essential for SARSCoV replication and transcription processes [42] ; <2> the genomic RNA produces two large proteins with overlapping sequences, polyproteins 1a and 1ab, which are autocatalytically cleaved by two or three viral proteases to yield functional polypeptides. The key enzyme in this processing is SARS 3CL protease [31] ; <2> a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160000 possible naturally occurring tetrapeptides. The enzyme exhibits a strong preference for P1 Gln containing substrates and P2 Leu containing substrates. The enzyme also shows a strong preference for P1 histidine containing substrates. 3Clpro has extended substrate specificity at P5 and P6 preferring hydrophobic amino acids such as Leu [6] ; <2> comprehensive overview of SARS-CoV 3CLpro substrate specificity. The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CLpro-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CLpro that is facing the P1 position is suggested to be occupied by acidic amino acids, thus the P1 position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or branching amino acids in P3 and P2 positions may also hinder the binding of SARS-CoV 3CLpro. In addition to the conserved Gln residue in the P1 position at the SARS-CoV 3CLpro cleavage site, the P2 position, which is exclusively occupied by Leu residue, also serves as another important determinant of substrate specificity. Peptide substrate with Phe replacement in the P2 position is also favorable for SARSCoV 3CLpro cleavage. Ile is intolerant in the P2 position. P1 position, which is frequently occupied by Ser residue, also contributes to the substrate specificity of SARS-CoV 3CLpro considerably. The P1 position is highly unfavorable to the substitution by Pro, Asp, and Glu residues. The substrate specificity of SARS-CoV 3CLpro is less dependent on the P2 and P3 positions at the cleavage site. The peptide cleavage results show that the P3 and P4 positions have no effect on determining the substrate specificity preferences of SARS-CoV 3CLpro [42] ; <2> cuts the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency [27] ; <2> the S3 subsite of the SARS CoVMpro has a negative character. The electrostatic interactions between Glu47 and P3Lys play a key role in specific binding. These observations are very important and provide further information for structural-based drug design against SARS virus [33] ; <2> 3CLpro cleaves the replicase polyproteins at 11 sites with the conserved Gln-(Ser, Ala, Gly) sequences [49] ; <2> no cleavage of SAVLQPGFRK [49] ) (Reversibility: ?) [6, 7, 27, 31, 33, 42, 49 ] P ?

(S)-2-((2S,3R)-2-((S)-2-acetamido-3-methylbutanamido)-3-(benzyloxy)butanamido)-4-methyl-N-((S)-4-(5-nitro-1,4-dioxo-3,4-dihydrophthalazin-2(1H)yl)-3-oxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)pentanamide <2> [15] 1,1'-sulfonylbis(4-nitrobenzene) <2> [21] 1-(1-benzothiophen-2-ylmethyl)-5-iodo-1H-indole-2,3-dione <2> [15, 16] 1-(2-naphthlmethyl)isatin-5-carboxamide <2> [50] 1-(2-naphthylmethyl)-2,3-dioxoindoline-5-carboxamide <2> [15] 1-[(1H-benzimidazol-5-ylcarbonyl)oxy]-1H-1,2,3-benzotriazole <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C 14 5A mutant enzyme [13] ) [13] 1-[(1H-indol-2-ylcarbonyl)oxy]-1H-1,2,3-benzotriazole <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C145A mutant enzyme [13] ) [13] 1-[(1H-indol-5-ylcarbonyl)oxy]-1H-1,2,3-benzotriazole <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C145A mutant enzyme [13] ) [13] 1

2-(benzylsulfanyl)-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile <2> [46] 2-[(1H-1,2,3-benzotriazol-1-yloxy)carbonyl]aniline <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C145A mutant enzyme [13] ) [13] 2 

inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C145A mutant enzyme [13] ) [13] 4-[(1H-1,2,3-benzotriazol-1-yloxy)carbonyl]-N,N-dimethylaniline <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C145A mutant enzyme [13] ) [13] 4-[(1H-1,2,3-benzotriazol-1-yloxy)carbonyl]-N-methylaniline <2> (<2> inhibition and irreversible mechanism-based inactivators, no irreversible inactivation with the C 14 5A mutant enzyme [13] ) [13] DTT <2> (<2> 80% of enzyme activity inhibited in the presence of 2.5 mM DTT [3] ) [3] Hg 2+ <2> [16] N-(2,2'-bithien-5-ylmethyl)-1,3-dimethyl-4-nitro-1H-pyrazol-5-amine <2> [21] N-(2-allylthiophenyl)cinnamide <2> [35] N- acetyl-Ala-Val-Leu-NHCH(CH 2 CH 2 CON(CH 3 ) 2 )-CHO <2> [31] acetyl-Ser-Ala-Val-Leu-NHCH(CH 2 CH 2 CON(CH 3 ) 2 )-CHO <2> [31] acetyl-Thr-Ser-Ala-Val-Leu-NHCH(CH 2 CH 2 CON(CH 3 ) 2 )-CHO <2> [31] acetyl-Val-Leu-NHCH(CH 2 CH 2 CON(CH 3 ) 2 )-CHO <2> [31] acetyl-leucylalanyl-alanyl-ketomethyl(cycloglutamine)-phthalhydrazide <2> [24] acetyl-valyl-(O-benzyloxy)threonyl-leucyl-ketomethyl(cycloglutamine)phthalhydrazide <2> [24] benzyl (2S,3S)-3-tert-butoxy-1-((S)-3-cyclohexyl-1-oxo-1-((S)-1-oxo-3-((S)-2oxopyrrolidin-3-yl)propan-2-ylamino)propan-2-ylamino)-1-oxobutan-2-ylcarbamate <2> [20] benzyl [(1S)-1-benzyl-3-chloro-2-oxopropyl]carbamate <2> (<2> potent and selective inhibitor [34] ) [34] benzyl [(1S)-3-chloro-1-(4-fluorobenzyl)-2-oxopropyl]carbamate <2> (<2> potent and selective inhibitor [34] ) [34] benzyl [(1S)-3-chloro-1-(naphthalen-2-ylmethyl)-2-oxopropyl]carbamate <2> (<2> potent and selective inhibitor [34] ) [34] benzyl [(1S,4S,7S,8R,9R,10S,13S,16S)-7,10-dibenzyl-8,9-dihydroxy-1,16-dimethyl-4,13-bis(1-methylethyl)-2,5,12,15,18-pentaoxo-20-phenyl-19-oxa-3,6,11,14,17-pentaazaicos-1-yl]carbamate <2> [16] benzyl [44] benzyl [(7S,8R,9R,10S)-8,9-dihydroxy-7,10-bis(1H-indol-3-ylmethyl)-1,16-dimethyl-4,13-bis(1-methylethyl)-2,5,12,15,18-pentaoxo-20-phenyl-19-oxa-3,6,11,14,17-pentaazaicos-1-yl]carbamate <2> (<2> highly selective for the 3CL protease and that no inhibitionis observed against HIV protease at 0.1 mM [14] ) [14] betulinic acid <2> (<2> competitive [22] ) [22] bis [1, 3] thiazolo[4,5-b:5',4'-e]pyridine-2,6-diamine <2> [15, 16] celastrol <2> (<2> competitive inhibitor [45] ) [45] cinaserin <2> [15, 35] curcumin <2> [22, 45] diethyl (2E,2'E)-3,3'-[sulfonylbis(benzene-4,1-diylimino)]bis(2-cyanoprop-2enoate) <2> [21] dihydrocelastrol <2> [45] esculetin-4-carboxylic acid ethyl ester <2> (<2> a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata [8] ) [ 

opyrrolidin-3-yl]pent-2-enoate <2> [11] extracts of Rheum palmatum <2> [48] hexachlorophene <2> [15, 16] iguesterin <2> (<2> competitive inhibitor [45] ) [45] methyl 3-([N-[(benzyloxy)carbonyl]-l-valyl]amino)-5-fluoro-4-oxopentanoate <2> (<2> potent and selective inhibitor [34] ) [34] methyl 4-hydroxy-6-(trifluoromethyl)furo[2,3-b]pyridine-2-carboxylate <2> [15, 16] niclosamide <2> [22] phenylmercuric acetate <2> [16] phenylmercuric nitrate <2> [16] pristimerin <2> (<2> competitive inhibitor [45] ) [45] savinin <2> (<2> competitive [22] ) [22] sulfonyldi-4,1-phenylene bis(2,3,3-trichloroacrylate) <2> [21] tert-butyl (3S)-3-[[(benzyloxy)carbonyl]amino]-5-bromo-4-oxopentanoate <2> (<2> potent and selective inhibitor [34] ) [34] tetraethyl 2,2'-[sulfonylbis(benzene-4,1-diyliminomethylylidene)]dipropanedioate <2> [21] thimerosal <2> [16] tingenone <2> (<2> competitive inhibitor [45] ) [45] toluene-3,4-dithiolato zinc <2> [16] zinc N-ethyl-N-phenyldithiocarbamate <2> [38] Additional information <2> (<2> molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S 1 pocket of SARS-CoV Mpro [10] ; <2> based on the X-ray structure of 3CLPro co-crystallized with a trans-a,b-unsaturated ethyl ester, a set of QM/QM and QM/MM calculations are performed, yielding three models with increasingly higher the number of atoms. It is suggested 3CLPro inhibitors need small polar groups to decrease the energy barrier for alkylation reaction. The results can be useful for the development of new compounds against SARS [39] ; <2> extracts from Rheum palmatum have a high level of inhibitory activity against 3CL protease with IC50 of 0.01376 mg/ml and an inhibition rate of up to 96% [48] ) [ [9] 0.27 <2> (Ser-Ala-Val-Leu-Gln-Met-Gly-Phe-Arg-Lys, <2> T25G mutant protein [49] ) [49] 0.31 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> E166A mutant protein [47] ) [47] 0.38 <2> (o-aminobenzoyl-TSAVLQSGFRY(NO2)G) [8] 0.48 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> R298L mutant protein [47] ) [47] 0.6 <2> (SITSAVLQ-p-nitrophenyl ester) [7] 0.63 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> wild-type protein [47] ) [47] 0.847 <2> (TFTRLQSLENV, <2> pH 7.3, room temperature [27] ) [27] 0.86 <2> (SITSAVLQ-p-nitroanilide) [ [20] 0.0007 <2> (phenylmercuric acetate) [16] 0.0008 <2> (iguesterin) [45] 0.001 <2> (N-ethyl-N-phenyldithiocarbamic acid zinc) [16] 0.0011 <2> (Zn 2+ ) [16] 0.0014 <2> (toluene-3,4-dithiolato zinc) [16] 0.0022 <2> (4,5-anhydro-2-([N-[(benzyloxy)carbonyl]-l-phenylalanyl]amino)-1,2-dideoxy-d-erythro-pent-3-ulose) [6] 0.0022 <2> (N-[(benzyloxy)carbonyl]-l-valyl-N-[(2S)-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]-1-(1,3-thiazol-2-yl)propan-2-yl]-l-leucinamide) [44] 0.00226 <2> (4-[2-(2-benzyloxycarbonylamino-3-methyl-butyrylamino)-3phenyl-propionylamino]-5-(2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester) [20] 0.0024 <2> (thimerosal) [16] 0.0031 <2> (pristimerin) [45] 0.004 <2> (tingenone) [45] 0.0042 <2> (celastrol) [45] 0.0045 <2> ([benzene-1,4-diylbis[oxycarbonyl(5-nitrobenzene-3,1-diyl)]]diboronic acid) [16] 0.006 <2> ([benzene-1,2-diylbis[methanediyloxycarbonyl(5-nitrobenzene-3,1diyl)]]diboronic acid) [16] 0.006 <2> ([benzene-1,3-diylbis[oxycarbonyl(5-nitrobenzene-3,1-diyl)]]diboronic acid) [16] 0.0082 <2> (betulinic acid) [22] 0.0091 <2> (savinin) [22] 0.0137 <2> (hexachlorophene) [15, 16] pH-Optimum 7 <2> (<2> substrate: TSAVLQSGFRK-NH 2 [27] ) [4, 27] 7.4 <2> (<2> wild-type enzyme [15] ) [15] 7.6 <2> (<2> mutant enzyme C145S [15] ) [15] 8 <2> [3] pH-Range 6-9 <2> (<2> pH 6: about 50% of maximal activity, pH 9: about 65% of maximal activity, substrate: TSAVLQSGFRK-NH 2 [27] ) [27] 6.3-8.7 <2> (<2> pH 6.3: about 55% of maximal activity, pH 8.7: about 45% of maximal activity, wild-type enzyme [15] ) [15] 6.3-9.3 <2> (<2> pH 6.3: about 55% of maximal activity, pH 9.3: about 45% of maximal activity, mutant enzyme C145S [15] ) [15] pi-Value 6.24 <2> (<2> mutant enzyme R188I, calculated from sequence [31] ) [31] Temperature optimum ( C) 42 <2> [3] 4 Enzyme Structure Molecular weight 33760 <2> (<2> determined by MALDI [3] ) [3] Subunits dimer <2> (<2> one monomer per asymmetric unit, dimer is generated through the crystallographic twofold [1] ; <2> by comparing molecular dynamics simulation of dimer and monomer, the indirect reasons for the inactivation of the monomer are found, that is the conformational variations of the active site in the monomer relative to dimer [25] ; <2> dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis [18] ; <2> the enzyme exists as a mixture of monomer and dimer at a higher protein concentration (4 mg/ml) and exclusively as a monomer at a lower protein concentration [16] ; <2> the SARS coronavirus main proteinase is a homodimer. Investigation of the monomer-dimer equilibrium [5] ; <2> a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml. The dimer may be the biological functional form of the protein [27] ; <2> in solution the wild type protease exhibits both forms of monomer and dimer and the amount of the monomer is almost equal to that of the dimeric form [37] ; <2> is only enzy-matically active as a homodimer. Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis [41] ; <2> SARS-CoV Mpro exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form [40] ; <2> wild type and D(300-306) proteases exist with dimer as the major form. The major form becomes monomeric in D(299-306), D(298-306) and D(297-306) [26] ) [1, 5, 16, 18, 25, 26, 27, 37, 40, 41] homodimer <2,3> (<2> X-ray crystallography [38] ; <2> analytical ultracentrifugation, gel filtration [47] ; <3> only the dimeric enzyme is active [51] ; <2> tendency of substrate induced dimer formation, gel filtration, analytic ultracentrifugation [50] ) [38, 47, 50, 51] monomer <2> (<2> by comparing molecular dynamics simulation of dimer and monomer, the indirect reasons for the inactivation of the monomer are found, that is the conformational variations of the active site in the monomer relative to dimer [25] ; <2> the enzyme exists as a mixture of monomer and dimer at a higher protein concentration (4 mg/ml) and exclusively as a monomer at a lower protein concentration [16] ; <2> a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ ml. The dimer may be the biological functional form of the protein [27] ; <2> in solution the wild type protease exhibits both forms of monomer and dimer and the amount of the monomer is almost equal to that of the dimeric form [37] ; <2> wild type and D(300-306) proteases exist with dimer as the major form. The major form becomes monomeric in D(299-306), D(298-306) and D(297-306) [26] ) [16, 25, 26, 27, 37] 

Purification <1> [19] <2> [6, 20, 21, 24, 27, 37] <2> (E166A mutant protein: immobilized metal ion affinity chromatography (Ni 2+ )) [47] <2> (Ni-NTA column chromatography) [49] <2> (ammonium sulfate precipitation and anion-exchange column chromatography) [48] <2> (ammonium sulfate precipitation, anion-exchange chromatography) [48] <2> (commercial preparation) [45] <2> (fused to maltose-binding protein, removing the maltose-binding protein by cleavage with factor Xa, purification by Phenyl Sepharose column chromatography) [1] <2> (glutathione S-transferase column chromatography and HiTrap 26/10 QFF column chroamtography) [38] <2> (immobilized metal ion affinity chromatography (Ni 2+ )) [49] <2> (purification of proteolysis-resistant mutant R188I of the SARS 3CL protease) [31] <2> (recombinant His-tagged SARS-CoV 3CL protease) [8] <2> (recombinant enzyme) [34] <2> (strong cation column chromatography connected in series to a strong anion column chromatography) [3] <2> (wild-type enzyme and C-terminally truncated proteases) [26] <3> (glutathione Sepharose column chromatography and Superdex 75 gel filtration) [51] Renaturation <2> (reversible unfolding of SARS-CoV main protease in guanidine-HCl. In the presence of 6 M of guanidine-HCl, the enzyme is completely unfolded in 10 min. The unfolding is completely reversible. A 10fold dilution induces refolding of the enzyme to a yield of 92-95%) [12] Crystallization <1> (crystal structure of monomeric mutant enzyme G11A) [19] <2> [21] <2> (1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor, an a,b-epoxyketone) [6] <2> (SARS 3CLpro bound to two phthalhydrazide-charged peptidyl inhibitors, acetyl-valyl-(O-benzyloxy)threonyl-leucyl-ketomethyl(cycloglutamine)phthalhydrazide and acetyl-leucylalanyl-alanyl-ketomethyl(cycloglutamine)phthalhydrazide. The inhibitors are covalently attached to SARS 3CLpro in crystals) [24] <2> (X-ray, resolution of 2.0 A for tetragonal crystals, 2.14 A for monoclinic crystals and 2.8 A for orthorhombic crystals, pH-dependent change of structure) [ [49] <2> (wild-type and mutant glutathione S-transferase-fusion constructs are transformed into Escherichia coli BL21 cell strain for overexpression) [17] <2> (wild-type enzyme and C-terminally truncated proteases, expression in Escherichia coli) [26] <3> (expressed in Escherichia coli BL21(DE3) cells) [51] Engineering C145A <2> (<2> no irreversible inactivation by benzotriazole esters [13] ) [13] C300A <2> (<2> mutant enzyme shows more than 30% of wild-type activity [17] ) [17] E14A <2> (<2> the ratio of dimer to monomer in solution is 0.36, compared to 1 for the wild-type enzyme [37] ) [37] E166A <2> (<2> involved in connecting the substrate binding site with the dimer interface, dimerization influenced by substrate binding [47] ) [47] E166A/R298A <2> (<2> monomer [47] ) [47] R298L <2> (<2> mutation destroys 85% of the enzyme activity [26] ; <2> induce dimer dissociation (influenced by substrate binding), about 10fold decrease in activty [47] ) [26, 47] R4A <2> (<2> the ratio of dimer to monomer in solution is 0.45, compared to 1 for the wild-type enzyme [37] ) [37] S10A <2> (<2> the ratio of dimer to monomer in solution is 0.66, compared to 1 for the wild-type enzyme [37] ) [37] S123A <2> (<2> mutation does not destroy the enzyme activity, the dimeric structure remains intact [26] ) [26] S123C <2> (<2> mutation does not destroy the enzyme activity, the dimeric structure remains intact [26] ) [26] S139A <2,3> (<2> mutation can destroy neither the enzyme activity nor the dimeric structure [26] ; <2> mutation does not destroy the enzyme activity, the dimeric structure remains intact [26] ; <2> the ratio of dimer to monomer in solution is 0.81, compared to 1 for the wild-type enzyme [37] ; <3> mutant S139A is a monomer that still retains a small fraction of dimer in solution, which may account for its remaining activity [51] ) [26, 37, 51] S1A <2> (<2> the ratio of dimer to monomer in solution is 1.08, compared to 1 for the wild-type enzyme [37] ) [37] S284A <2> (<2> activity is higher than that of wild-type enzyme [17] ) [17] S284A/T285A/I286A <2> (<2> activity is 3.7fold higher than wild-type activity [17] ) [17] S301A <2> (<2> no significant activity differences from the wild-type protease [17] ) [17] T25G <2> (<2> activity like wild-type (substrate DABCYL-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS), higher specific activity than wild-type protein for substrate Ser-Ala-Val-Leu-Gln-Met-Gly-Phe-Arg-Lys [49] ; <2> the mutant enzyme has an expanded S1 space that demonstrates 43.5-fold better k cat /K m compared with wild type in cleaving substrates with a larger Met at P1, mutant enzyme T25G shows a 12fold and 8fold higher activity against the substrates with Met and Leu at P1, respectively [49] ) [49] T25S <2> (<2> almost complete loss of activity [49] ) [49] T280A <2> (<2> no significant activity differences from the wild-type protease [17] ) [17] T285A <2> (<2> activity is higher than that of wild-type enzyme [17] ) [17] Additional information <2> (<2> truncation of C-terminus from 306 to 300 has no appreciable effect on the quaternary structure, and the enzyme remains catalytically active. Further deletion of Gln299 or Arg298 drastically decreases the enzyme activity to 1-2% of wild type, and the major form is a monomeric one. The catalytic constant and specificity constant (k cat /K m ) of the proteases are significantly decreased in the D(299-306), D(298-306), and D(297-306) mutants. Wild type and D(300-306) proteases exist with dimer as the major form. The major form becomes monomeric in D(299-306), D(298-306) and D(297-306) [26] ; <2> without the N-finger, SARS-CoV Mpro can no longer retain the active dimer structure. It can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV Mpro is not only cri-tical for its dimerization but also essential for the enzyme to form the enzymatically active dimer [40] ) [26, 40] Application analysis <2> (<2> developing a novel red-shifted fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms [8] ) [8] medicine <2> (<2> SARS-3CLpro is a viral cysteine protease critical to the life cycle of the pathogen and hence a therapeutic target of importance [29] ; <2> this enzyme is a target for the design of potential anti-SARS drugs [28] ) [28, 29] 6 Stability Temperature stability 61 <2> (<2> Tm-value, sigmoid denaturation curve [27] ) [27] Organic solvent stability guanidine-HCl <2> (<2> dimeric enzyme dissociates at guanidinium chloride concentration of less than 0.4 M, at which the enzymatic activity loss showes close correlation with the subunit dissociation. Further increase in guanidinium chloride induces a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme follows a monophasic unfolding curve. Unfolding curves of mutants of the full-length protease W31 and W207/W218 are monophasic but correspond to the first and second phases of the protease, respectively. The unfolding intermediate of the protease represents a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties [12] ) [12] General stability information <2>, replacing Arg with Ile at position 188 renders the protease resistant to proteolysis [31] 

3> (<2> the ratio of dimer to monomer in solution is 0.63, compared to 1 for the wild-type enzyme [37]; <3> mutant F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabiliz

G283A <2> (<2> no significant activity differences from the wild-type protease

L282A <2> (<2> mutant enzyme shows more than 30% of wild-type activity

N214A <2> (<2> mutant enzyme shows more than 30% of wild-type activity

N289A <2> (<2> mutant enzyme retains less than 10% of the wild-type activity

-dimethylaminophenylazo)benzoic acid]-KNSTLQSGLRKE-[5-[2-(aminoethyl)amino]-naphthalenesulfonic acid] is 1.14 fold lower than wild-type value, K m -value for the substrate

Q299A <2> (<2> mutant enzyme retains less than 10% of the wild-type activity

The catalytic ability of 3CL-R188I protease was found to be extreme as compared to that of a mature 3CL protease containing a Cterminal His tag. The k cat values is 0.0203 per sec for mature 3CL protease and 4753 per sec for the 3CL

R298A <2> (<2> mutant enzyme retains less than 10% of the wild-type activity

<2> monomeric mutant

<2> the quaternary structures of exists in a mixture of monomeric and dimeric forms [26]; <2> induce dimer dissociation (influenced by substrate binding), about 10fold decrease in activty

<2> mutant is present almost exclusively in the monomeric form

Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer

Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs

Enzymatic activity of the SARS coronavirus main proteinase dimer

pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses

SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant

Substrate specificity profiling and identification of a new class of inhibitor for the major protease of the SARS coronavirus

Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis

Development of a red-shifted fluorescence-based assay for SARScoronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata

Binding interaction of quercetin-3-b -galactoside and its synthetic derivatives with SARS-CoV 3CLpro: Structure-activity relationship studies reveal salient pharmacophore features

Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S 1 pocket of SARS-CoV Mpro

Structure-based design, synthesis, and biological evaluation of peptidomimetic SARS-CoV 3CLpro inhibitors

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease

Structure-based design and synthesis of highly potent SARS-CoV 3CL protease inhibitors

Quaternary structure, substrate selectivity and inhibitor design for SARS 3C-like proteinase

Characterization and inhibition of SARS-coronavirus main protease

The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain

Only one protomer is active in the dimer of SARS 3C-like proteinase

Mutation of Gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3C-like protease. Crystal structure with molecular dynamics simulations

Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease

Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus

Crystal structures reveal an induced-fit binding of a substrate-like aza-peptide epoxide to SARS coronavirus main peptidase

A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase

Insight into the activity of SARS main protease: molecular dynamics study of dimeric and monomeric form of enzyme

Correlation between dissociation and catalysis of SARS-CoV main protease

Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase

Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase

Structure-based virtual screening against SARS-3CL(pro) to identify novel non-peptidic hits

Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease

Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

Development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease 3CL(pro)

Design and synthesis of cinanserin analogs as severe acute respiratory syndrome coronavirus 3CL protease inhibitors

Individual and common inhibitors of coronavirus and picornavirus main proteases

Residues on the dimer interface of SARS coronavirus 3C-like protease: dimer stability characterization and enzyme catalytic activity analysis

Structural basis of inhibition specificities of 3C and 3C-like proteases by zinccoordinating and peptidomimetic Compounds

QM/QM studies for Michael reaction in coronavirus main protease (3CL Pro)

Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain

Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3C-like protease

Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

C-terminal domain of SARS-CoV main protease can form a 3D domain-swapped dimer

New developments for the design, synthesis and biological evaluation of potent SARS-CoV 3CL(pro) inhibitors

SARS-CoV 3CLpro inhibitory effects of quinonemethide triterpenes from Tripterygium regelii

Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease

Mutation of Glu-166 blocks the substrate-induced dimerization of SARS coronavirus main protease

Anti-SARS coronavirus 3C-like protease effects of Rheum palmatum L. extracts

Engineering a novel endopeptidase based on SARS 3CL(pro)

Maturation mechanism of severe acute respiratory syndrome (SARS) coronavirus 3C-like proteinase

Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure