No abstract

An artificial tetracontapeptide that mimicked the recognition/binding properties of naturally occurring opioid receptors was designed, synthesized, and purified to homogeneity. The design of the primary structure of the receptor mimetic peptide (RMP) accounted for secondary structure prediction rules and for the stereochemical anatomy of various enkephalin and morphine derivatives. The affinity of a series of opioid and nonopioid peptides to RMP was determined from their potency in displacing the binding of enzymatically prepared (14C)-[Leu]-enkephalin. The competition studies revealed that the binding is specific for endogenous opiate peptides, stereoselective for the naturally occurring L isomer of [Leu]-enkephalin, and discriminative for closely related opioid peptides. The thermodynamic parameters associated with the binding of [Leu]-enkephalin to RMP were evaluated from equilibrium studies at different temperatures. The van't Hoff plot of the resulting data was curvilinear. The formation of the ligand--RMP complex was characterized by a decrease both in entropy and in enthalpy with temperature. The thermodynamic behavior provided some evidence that hydrophobic interactions played a prominent role in stabilizing the [Leu]-enkephalin--RMP complex.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. Na-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of oa-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin-and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.

No abstract