New substrate-derived thiol-specific time-dependent inhibitors for the characterisation of the coupling of binding site interactions with catalytic site chemistry in the cysteine proteinases

1993 ◽  
Vol 21 (2) ◽  
pp. 215S-215S
Author(s):  
Geoffrey W. MELLOR ◽  
Manij PATEL ◽  
Mark P. THOMAS ◽  
Devanand KOWLESSUR ◽  
Suneal K. SREEDHARAN ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Site ◽  
Time Dependent ◽  
Cysteine Proteinases ◽  
Specific Time

Characterization of binding site-catalytic site signalling in cysteine proteinases by using substrate-derived two-protonic-state reactivity probes

1986 ◽  
Vol 14 (6) ◽  
pp. 1225-1226 ◽  
Author(s):  
KEITH BROCKLEHURST ◽  
DEVANAND KOWLESSUR ◽  
GEETA PATEL ◽  
WILLIAM TEMPLETON ◽  
EMRYS THOMAS ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Site ◽  
Cysteine Proteinases

scholarly journals Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding

1987 ◽  
Vol 244 (1) ◽  
pp. 173-181 ◽  
Author(s):  
K Brocklehurst ◽  
D Kowlessur ◽  
M O'Driscoll ◽  
G Patel ◽  
S Quenby ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Ph Dependence ◽  
Catalytic Site ◽  
Amide Bond ◽  
Side Chain ◽  
Specific Substrate ◽  
Cysteine Proteinases ◽  
Pyridyl Nitrogen ◽  
Compound Ii

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2′-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2′-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Ficin: a cysteine proteinase with binding site-catalytic site signalling characteristics intermediate between those of papain and actinidin

1993 ◽  
Vol 21 (2) ◽  
pp. 216S-216S
Author(s):  
Manij PATEL ◽  
Mark P. THOMAS ◽  
Irfan S. KAYANI ◽  
Geoffrey W. MELLOR ◽  
Emrys W. THOMAS ◽  
...  
Keyword(s):  
Binding Site ◽  
Cysteine Proteinase ◽  
Catalytic Site

scholarly journals A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

2020 ◽  
Vol 295 (28) ◽  
pp. 9474-9489 ◽  
Author(s):  
Manon Molina ◽  
Claire Moulis ◽  
Nelly Monties ◽  
David Guieysse ◽  
Sandrine Morel ◽  
...  
Keyword(s):  
Binding Site ◽  
Molar Mass ◽  
Enzyme Stability ◽  
Catalytic Site ◽  
Glycoside Hydrolase Family ◽  
Surface Binding ◽  
High Molar Mass ◽  
Sugar Binding ◽  
Binding Pockets ◽  
Domain V

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

scholarly journals Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels

2008 ◽  
Vol 295 (2) ◽  
pp. C557-C565 ◽  
Author(s):  
Sriharsha Vemana ◽  
Shilpi Pandey ◽  
H. Peter Larsson
Keyword(s):  
Binding Site ◽  
Physiological Role ◽  
Time Dependent ◽  
Inward Rectification ◽  
Hcn Channels ◽  
Membrane Hyperpolarization ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Hcn1 Channels ◽  
Pore Blocker

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents ( 5 ). In the present study, we show that intracellular Mg2+ functions as a voltage-dependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg2+ is in the physiological range, with Mg2+ binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at +50 mV. The effective electrical distance for the Mg2+ binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg2+, suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg2+ acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg2+ may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg2+ could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.

A dual-level state-specific time-dependent density-functional theory

2008 ◽  
Vol 29 (8) ◽  
pp. 1187-1197 ◽  
Author(s):  
Seiken Tokura ◽  
Takeshi Sato ◽  
Takao Tsuneda ◽  
Takahito Nakajima ◽  
Kimihiko Hirao
Keyword(s):  
Density Functional Theory ◽  
Density Functional ◽  
Time Dependent ◽  
Specific Time ◽  
Functional Theory ◽  
Dependent Density

scholarly journals Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression

2005 ◽  
Vol 52 (8) ◽  
pp. 2386-2395 ◽  
Author(s):  
Jennifer H. Lee ◽  
Jonathan B. Fitzgerald ◽  
Michael A. DiMicco ◽  
Alan J. Grodzinsky
Keyword(s):  
Gene Expression ◽  
Mechanical Injury ◽  
Time Dependent ◽  
Cartilage Explants ◽  
Specific Time
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