Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding.
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)
Brocklehurst
K
K
Department of Biochemistry, Medical College of St. Bartholomew's Hospital, University of London, U.K.
Kowlessur
D
D
O'Driscoll
M
M
Patel
G
G
Quenby
S
S
Salih
E
E
Templeton
W
W
Thomas
E W
EW
Willenbrock
F
F
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000378:metabolism; D000081:Acetamides / Q000378:metabolism*; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004220:Disulfides; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000081:Acetamides; D004220:Disulfides; D011725:Pyridines; D013438:Sulfhydryl Compounds; C053806:2-(acetamido)ethyl-2'-pyridyl disulfide; C001105:2,2'-dipyridyl disulfide; D010649:Phenylalanine; D015082:2,2'-Dipyridyl; C016698:n-propyl 2-pyridyl disulfide; C053807:2-(N'-acetylphenylalanylamino)ethyl-2'-pyridyl disulfide; D003546:Cysteine Endopeptidases