Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Figures & data

Scheme 1.  Reaction schemes for irreversible inhibition (according to Ref. 3).

Scheme 2.  Proposed mechanism of inhibition of serine hydrolases by 3,4-dichloroisocoumarin. Adapted from Ref. 5.

Figure 1.  Time-course of SrLip–DCI complex reactivation after incubation of 4.55 µM Streptomyces rimosus lipase with the 10-fold molar excess of DCI, followed by activity assay and mass spectrometric (MALDI-MS and ESI-MS) measurements. Curve fitting performed in GraphPad Prism 4.0. DCI, 3,4-dichloroisocoumarin; ESI, electro-spray ionization; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; SrLip, Streptomyces rimosus lipase; TOF, time-of-flight.

Figure 2.  Time-course of SrLip–DCI complex reactivation after incubation of 4.55 µM Streptomyces rimosus lipase with the 10-fold molar excess of DCI, followed by MALDI-TOF-MS. Incubation times are indicated on the right. Only region encompassing doubly protonated ions is shown. DCI, 3,4-dichloroisocoumarin; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; SrLip, Streptomyces rimosus lipase.

Table 1.  Half-lives and zero-order constants for the spontaneous hydrolysis of DCI in different buffer systems.

Buffer	k (min^−1)	t1/2 (min)	ϵ325 (M^−1 cm^−1)
0.1 M HEPES with 0.5 M NaCl and 10% DMSO pH 7.5	0.0385^a	18.0^Citation5	3330.0^Citation5
0.02 M K2HPO4 with 0.15 M NaCl and 10% DMSO pH 7.4	0.0144^a	48.0^Citation5
10 mM Na-PO4 with 0.2 M NaCl and 10 mM EDTA and 1%DMSO pH 6.8 (buffer 2)	0.0004	121.2	3794.7
50 mM Na-PO4 with 5% DMSO pH 8.0 (buffer 1)	0.0029	35.8	3299.4

EDTA, ethylenediaminetetraacetic acid.

^a First-order constants, calculated from t_1/2⁵.

Table 2.  Inhibition of serine proteases and other enzymes by 3,4-dichloroisocoumarin.

Enzyme	[DCI] (µM)	t1/2 (min)	kobs/[I] (M^−1 s^−1)
Serine proteases
Human leukocyte elastase^Citation5	1.1	1.2	8920
Human leukocyte elastase^Citation44	0.6–6.0		12,800
Murine granzyme B^Citation6	4.2		4200
Staphylococcus aureus protease V-8^Citation5	18.0	0.3	2765
Human proteinase 3^Citation6	3.6		2600
Human proteinase 3^Citation44	0.6–6.0		6389
Porcine pancreatic elastase^Citation5	8.1	0.6	2500
Rat mast cell protease II^Citation5	11.0	1.8	580
Bovine chymotrypsin Aα^Citation5	13.0	1.6	570
Streptomyces griseus protease A^Citation5	136.0	0.3	310
Rat mast cell protease I^Citation5	38.0	1.2	260
Bovine trypsin^Citation5	127.0	0.5	198
Human factor D^Citation5	109.0	0.6	192
Human factor D^Citation45	50.0		40
Human C1s^Citation6	44.0		170
Trypanosoma congolense trypsin-like serineoligopeptidase^Citation46	250.0	0.3	167
Human plasmin^Citation5	203.0	0.4	133
Human factor XIIa^Citation5	135.0	1.3	64
Murine granzyme A^Citation6	45.0		50
Human C1r^Citation6	470.0		42
Sheep lymph capillary CIP^Citation6	460.0		39
Human factor VIIa^Citation6	44.0		31
Protease La^Citation6	82.0		30
Human leukocyte cathepsin G^Citation5	49.0	8.4	28
Human skin chymase^Citation5	92.0	4.7	27
Porcine pancreatic kallikrein^Citation5	127.0	3.4	27
Bovine factor XIa^Citation5	239.0	1.8	27
Bovine thrombin^Citation5	127.0	3.7	25
Human thrombin^Citation5	340.0	3.4	10
Bovine factor Xa^Citation5	422.0	133.0	0.2
Dipeptidyl-peptidase IV^Citation6	50.0		18%
Esterases and other enzymes
Streptomyces rimosus lipase	1.1–13.8	0.1–1.0	5705
Influenza C virus esterase^Citation6	6.3		410
Bovine multicatalytic proteinase complex^Citation6
Chymotrypsin-like activity	4.0		147
Glutamyl-hydrolyzing activity	12.0		33
Trypsin-like activity	40.0		12
Rat liver multicatalytic proteinase complex^Citation47
Chymotrypsin-like activity	20.0	4.8	120
Peptidylglutamylpeptide hydrolase activity-cooperative component	20.0	12.0	48
Glycogen phosphorylase b^Citation6	100.0		3.4
Acetylcholinesterase^Citation5	157.0	>120	<0.6

Bold numbers represent the values obtained in this study.

Figure 3.  View of the bound inhibitor in the structure of the complex of human factor D with DCI (PDB code 1dic). a) The DCI molecule is covalently bound to the catalytic Ser-195, whereas the catalytic residues His-57 and Asp-102 are out of the pocket with the bound inhibitor. Two hydrogen bond contacts between factor D and DCI are found: a carboxyl oxygen of inhibitor acts as the acceptor to nitrogen of Gly-193 (N–H…O, 2.73 Å) and the carbonyl oxygen is in a weak contact to Gly-216 (3.44 Å). b) The same orientation of the inhibitor depicted against the surface of the protein shaded according to the hydrophobicity of the residues (light to dark with increasing hydrophobicity). DCI, 3,4-dichloroisocoumarin.

Figure 4.  Inhibition of Streptomyces rimosus lipase by 3,4-dichloroisocoumarin: a) incubation method, b) progress curve method.

Figure 5.  Titration of 3 µM SrLip with DCI, followed by ESI-MS after 15 min of incubation of enzyme with increasing concentration of inhibitor. DCI, 3,4-dichloroisocoumarin; ESI, electro-spray ionization; MS, mass spectrometry; SrLip, Streptomyces rimosus lipase.

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