No abstract

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a 'featureless' probe), 2-(acetamido)ethyl 2'-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2'-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2'-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

A new, automated, knowledge‐based method for the construction of three‐dimensional models of proteins is described. Geometric restraints on target structures are calculated from a consideration of homologous template structures and the wider knowledge base of unrelated protein structures. Three‐dimensional structures are calculated from initial partly folded states by high‐temperature molecular dynamics simulations followed by slow cooling of the system (simulated annealing) using nonphysical potentials. Three‐dimensional models for the biotinylated domain from the pyruvate carboxylase of yeast and the lipoylated H‐protein from the glycine cleavage system of pea leaf were constructed, based on the known structures of two lipoylated domains of 2‐oxo acid dehydrogenase multienzyme complexes. Despite their weak sequence similarity, the three proteins are predicted to have similar three‐dimensional structures, representative of a new protein module. Implications for the mechanisms of posttranslational modification of these proteins and their catalytic function are discussed.