Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.
1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)
Brocklehurst
K
K
Department of Biochemistry, St. Bartholomew's Hospital Medical College, University of London, U.K.
Kowlessur
D
D
Patel
G
G
Templeton
W
W
Quigley
K
K
Thomas
E W
EW
Wharton
C W
CW
Willenbrock
F
F
Szawelski
R J
RJ
D000081:Acetamides / Q000378:metabolism*; D000577:Amides; D001562:Benzimidazoles / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D010206:Papain / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D011725:Pyridines / Q000378:metabolism*; D012997:Solvents; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000081:Acetamides; D000577:Amides; D001562:Benzimidazoles; D046911:Macromolecular Substances; D011522:Protons; D011725:Pyridines; D012997:Solvents; C051672:2-ethylpyridine; C053806:2-(acetamido)ethyl-2'-pyridyl disulfide; C056421:benzimidazol-2-ylmethanethiol; D003903:Deuterium; D010206:Papain