LSpI

Linear specific inhibition

Fingerprints of LSpI


#Enzyme
Species
EC no.ModifierSubstrate(1)Name given by authors(2)Reference (3)
1Alpha,alpha trehalase
Chironomus riparius
3.2.1.287-deoxy-epi-casuarine
(Compound 6)
TrehalosePure competitive inhibition
KX = 118 nM
D'Adamio
(2015)
2Coagulation factor IXa
Homo sapiens
3.4.21.27AprotininL-Pyroglutamyl-L-prolyl-L-arginine-p-nitroanilideCompetitive inhibition
KX = 1.23 μM
Ogawa
(2005)
3Cholinesterase (4)
Homo sapiens
3.1.1.8Tosyllysine chloromethyl ketoneButyrylthiocholinePure competitive inhibition
KX = 8 μM
Çokuğraş
(2004)
4Aldehyde oxidase
Homo sapiens
1.2.3.16-ChloroquinazolinonePhtalazineStrictly competitive inhibition
KX = 5.4 μM
Barr
(2011)
5Spermine synthase
Bos taurus
2.5.1.22Spermine
(reaction product)
Decarboxylated adenosylmethionineCompetitive inhibition
KX ≈ 0.028 μM (5)
Pajula
(1983) (6)
6Tyrosinase (7)
Homo sapiens
1.14.18.1HTS05357L-DopaPurely competitive inhibition
KX = 24.2 μM
Sun
(2014) (8)
7Dipeptidyl-peptidase IV
Homo sapiens
3.4.14.5Trp2,Ile3-Tat(1–9)
MWIVDPNIE
Ala-Pro-p-nitroanilideCompetitive inhibition
KX = 43.6 μM
Lorey
(2003) (9)
8Dipeptidyl-peptidase IV
Homo sapiens
3.4.14.5TXA2-R(1–9)
MWPNGSSLG
Ala-Pro-p-nitroanilideCompetitive inhibition
KX = 5.02 μM
Lorey
(2003) (9)
9Dipeptidyl-peptidase IV
Homo sapiens
3.4.14.5Met-Trp1-IL-2(1–8)
MWPTSSSTK
Ala-Pro-p-nitroanilideCompetitive inhibition
KX = 15.9 μM
Lorey
(2003) (9)
10Dipeptidyl-peptidase IV
Homo sapiens
3.4.14.5Met-Trp-Pro
MWP
Ala-Pro-p-nitroanilideCompetitive inhibition
KX = 24.5 μM
Lorey
(2003) (9)
11Acetylcholinesterase
Torpedo californica
3.1.1.7Edrophonium7-Acetoxy-4-methylcoumarinLinear competitive inhibition
KX = 0.14 μM
Berman
(1990)
12Acetylcholinesterase
Torpedo californica
3.1.1.7Phenyltrimethylammonium7-Acetoxy-4-methylcoumarinLinear competitive inhibition
KX = 17 μM
Berman
(1990)
13Acetylcholinesterase
Torpedo californica
3.1.1.7N-Methylacridinium7-Acetoxy-4-methylcoumarinLinear competitive inhibition
KX = 0.11 μM
Berman
(1990)
14Gamma-glutamyltransferase
Homo sapiens
2.3.2.2OU749Gly-Gly (10)Competitive inhibition
KX = 17.6 μM
King
(2009)
15Thrombin
Homo sapiens
3.4.21.5Recombinant hirudinTos-Gly-Pro-Arg-7-amino-4-methyl-
coumarylamide
Competitive inhibition
KX= 447 fM
Schmitz
(1991)
16Thrombin
Homo sapiens
3.4.21.5Hirudin1-47 fragmentTos-Gly-Pro-Arg-7-amino-4-methyl-
coumarylamide
Competitive inhibition
KX= 0.42 μM
Schmitz
(1991)
17Thrombin
Homo sapiens
3.4.21.5Hirudin45-65 fragmentFibrinogen AαCompetitive inhibition
KX= 0.76 μM
Schmitz
(1991)
18Thrombin
Homo sapiens
3.4.21.5Hirudin1-47 fragmentFibrinogen AαCompetitive inhibition
KX= 0.46 μM
Schmitz
(1991)
19Protein-lysine desuccinylase (NAD+)
Homo sapiens
2.4.2.B14Compound 39Abz-
GVLK(glutaryl)AY(NO2)GV-NH2
Competitive inhibition
KX= 83.2 nM
Kalbas (11)
(2018)
20Protein-lysine desuccinylase (NAD+)
Homo sapiens
2.4.2.B14Compound 39.2Abz-
GVLK(glutaryl)AY(NO2)GV-NH2
Competitive inhibition
KX= 13.6 nM
Kalbas (11)
(2018)
21Coagulation factor Xa
Homo sapiens
3.4.21.6BAY 59-7939
(Rivaroxaban)
N-Methoxycarbonyl-D-cyclohexylalanyl-
glycyl-L-arginine-4-nitroanilide acetate
Competitive inhibition
KX= 0.4 nM
Perzborn
(2005)
22Acylglycerol lipase
Homo sapiens
3.1.1.23Compound 17b4-nitrophenylacetateCompetitive inhibition
KX= 0.65 μM
Granchi
(2016)
23ABC-type quaternary amine transporter
Mus musculus
7.6.2.9Compound 9γ-Aminobutyric acidCompetitive inhibitionAl-Khawaja
(2014)
24beta-Galactosidase
Escherichia coli
3.2.1.23β-D-1-thiogalacto-
pyranoside (IPTG)
Resorufin β-D-galactopyranosideCompetitive inhibition
KX= 63 μM
Hess (12)
(2015)
253-Hydroxyacyl-[acyl-carrier-protein] dehydratase
Yersinia pestis
4.2.1.59MangostinCrotonyl-coenzyme-ACompetitive inhibition
KX= 1.4 μM
McGillick
(2016)
26Caspase-3
Homo sapiens
3.4.22.56Designed ankyrin repeat protein D3.4N-Acetyl-DEVD-7-amino-4-methylcoumarylamidePurely competitive inhibition
KX= 16.8 nM
Schroeder
(2013)

(1) Always the varied substrate. In two- or more-substrate reactions the concentration(s) of the non varied substrate(s) is/are kept constant.

(2) Name of the mechanism given by the authors in the quoted reference. α, β and the inhibition/activation constants for the modifier (X), uniformly denoted KX, are the values specified by the authors. In some cases,  missing parameters have been calculated from graphical or tabular data provided in the papers. In two- or more-substrate reactions, KX represents an apparent constant at given concentrations of the fixed substrates and no calculations of the intrinsic values have been attempted.

(3) Full references at the end of the page provide also the digital object identifier (doi), if available. Clicking the authors (highlighted) opens the reference in PubMed.

(4) Heat desensitized enzyme, 24 h at 45°C.

(5) Approximate value calculated from the graphic in Fig. 5.

(6) A study dedicated to inhibition of spermine synthase (two substrates-two products reaction) by reaction products that suggests a compulsory-order mechanism in which both substrates bind the enzyme before release of products. See also the pages of LMx(Sp>Ca)I and LMx(Sp<Ca)I.

(7) In this study a truncated, His-tagged form of tyrosinase, which includes the catalytic domain, was used.

(8) This paper by Sun and coworkers is a recommended reading because it is a useful tutorial in the management of enzyme-inhibition kinetic data. The authors fit the general modifier mechanism model of Botts and Morales to an entire ensemble of data with a large number of experimental points. The analysis of inhibition mechanism is based on graphical representation of data and on statistical test that include the Akaike information criterion. See also results from this study under LMx(Sp>Ca)I and LMx(Sp<Ca)I.

(9) The study by Lorey  et al. (2003) is centered on the inhibition of human dipeptidyl-peptidase IV by the peptide MDPVDPNIE, Tat(1-9), corresponding to the N-terminus of the human immunodeficiency virus-1 transactivator Tat (HIV-1 Tat, which consists of 86 amino acids), and derived peptides. See also cases of linear mixed, predominantly specific inhibition, with/without multiple inhibitor binding: LMx(Sp>Ca)I.

(10) Transpeptidation reaction of gamma-glutamyltransferase:  (5-L-glutamyl)-peptide + amino acid = peptide + 5-L-glutamyl amino acid. See also LCaI.

(11) A synonym of Protein-lysine desuccinylase is Sirtuin 5. This is one of few papers in which the kinetic mechanism, LSpI, has been accurately demonstrated by showing the linear dependence of Kmapp on modifier concentration for twelve compounds analyzed in every due detail (paper and Supplement).

(12) The authors describe the innovative method Stroboscopic Epifluorescence Imaging  for investigating enzyme kinetics, which allows ‘high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes’. This must-read paper holds the record number of data points in a single experiment!


References

  1. Al-Khawaja A, Petersen JG, Damgaard M, Jensen MH, Vogensen SB, Lie MEK, Kragholm B, Bräuner-Osborne H, Clausen RP, Frølund B, Wellendorph P (2014) Pharmacological identification of a guanidine-containing β-alanine analogue with low micromolar potency and selectivity for the betaine/GABA transporter 1 (BGT1). Neurochem Res 39: 1988-1996. doi:10.1007/s11064-014-1336-9
  2. Barr JT, Jones JP (2011) Inhibition of human liver aldehyde oxidase: implications for potential drug-drug interactions. Drug Metab Disp 39: 2381-2386. doi:10.1124/dmd.111.041806
  3. Berman HA, Leonard K (1990) Ligand exclusion on acetylcholinesterase. Biochemistry 29: 10640-10649. doi:10.1021/bi00499a010
  4. Çokuğraş AN, Cengiz D, Tezcan EF (2004) Do alkylating agents modify the histidine residue of the desensitized butyrylcholinesterase? Protein J 23: 495-500. doi:10.1007/s10930-004-7876-0
  5. D’Adamio G, Sgambato A, Forcella M, Caccia S, Parmeggiani C, Casartelli M, Parenti P, Bini D, Cipolla L, Fusi P, Cardona F (2015) New synthesis and biological evaluation of uniflorine A derivatives: towards specific insect trehalase inhibitors. Org Biomol Chem 13: 886-892. doi:10.1039/C4OB02016B
  6. Granchi C, Rizzolio F, Palazzolo S, Carmignani S, Macchia M, Saccomanni G, Manera C, Martinelli A, Minutolo F, Tuccinardi T (2016) Structural optimization of 4-chlorobenzoylpiperidine derivatives for the development of potent, reversible, and selective monoacylglycerol lipase (MAGL) inhibitors. J Med Chem 59: 10299-10314. doi:10.1021/acs.jmedchem.6b01459
  7. Hess D, Rane A, deMello AJ, Stavrakis S (2015) High-throughput, quantitative enzyme kinetic analysis in microdroplets using stroboscopic epifluorescence imaging. Anal Chem 87: 4965-4972. doi:10.1021/acs.analchem.5b00766
  8. Kalbas D, Liebscher S, Nowak T, Meleshin M, Pannek M, Popp C, Alhalabi Z, Bordusa F, Sippl W, Steegborn C, Schutkowski M (2018) Potent and selective inhibitors of human sirtuin 5. J Med Chem 61: 2460-2471. doi:10.1021/acs.jmedchem.7b01648
  9. King JB, West MB, Cook PF, Hanigan MH (2009) A novel, species-specific class of uncompetitive inhibitors of gamma-glutamyl transpeptidase. J Biol Chem 284: 9059-9065. doi:10.1074/jbc.M809608200
  10. Lorey S, Stöckel-Maschek A, Faust J, Brandt W, Stiebitz B, Gorrell MD, Kähne T, Mrestani-Klaus C, Wrenger S, Reinhold D, Ansorge S, Neubert K (2003) Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites. Eur J Biochem 270: 2147-2156. doi:10.1046/j.1432-1033.2003.03568.x
  11. McGillick BE, Kumaran D, Vieni C, Swaminathan S (2016) β-Hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: structure determination, enzymatic characterization, and cross-inhibition studies. Biochemistry 55: 1091-1099. doi:10.1021/acs.biochem.5b00832
  12. Ogawa T, Verhamme IM, Sun MF, Bock PE, Gailani D (2005) Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX. J Biol Chem 280: 23523-23530. doi:10.1074/jbc.M500894200
  13. Pajula RL (1983) Kinetic properties of spermine synthase from bovine brain. Biochem J 215: 669-676. doi:10.1042/bj2150669
  14. Perzborn E, Strassburger J, Wilmen A, Pohlmann J, Roehrig S, K.H. S, Straub A (2005) In vitro and in vivo studies of the novel antithrombotic agent BAY 59‐7939—an oral, direct Factor Xa inhibitor. J Thromb Haemost 3: 514-521. doi:10.1111/j.1538-7836.2005.01166.x
  15. Schmitz T, Rothe M, Dodt J (1991) Mechanism of the inhibition of α-thrombin by hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Eur J Biochem 195: 251-256. doi:10.1111/j.1432-1033.1991.tb15701.x
  16. Schroeder T, Barandun J, Fluetsch A, Briand C, Mittl PRE, Gruetter MG (2013) Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors. Structure 21: 277-289. doi:10.1016/j.str.2012.12.011
  17. Sun W, Wendt M, Klebe G, Röhm KH (2014) On the interpretation of tyrosinase inhibition kinetics. J Enzyme Inhib Med Chem 29: 92-99. doi:10.3109/14756366.2012.755621