N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitory properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoylaminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.Papain-catalysed hydrolyses proceed by a reaction pathway which involves minimally three steps :where ES is the Michaelis complex, ES' is the acylenzyme formed through the thiol group of cysteine-25, Pz is the acid and PI the alcohol or amine moeity of the hydrolysed substrate. There is, however, indirect evidence for the involvement of tetrahedral intermediates in the acylation step, kz, and if established this would make a tetrahedral intermediate in the deacylation step, k3, virtually mandatory [l -31.The specificity of papain for structural features in the acyl moiety of the substrate manifests itself very largely in the formation of the acyl-enzyme from the enzyme-substrate complex, i.e. the specificity is largely observed in the acylation rate constant, kz [4]. This feature of kinetic specificity is common amongst the proteolytic enzymes and in principle can arise from three distinct causes : non-productive binding, a substrate-induced conformational change in the enzyme, or substrate destabilisation in the enzyme-substrate complex [5]. Model building suggested that a non-bonded interaction between the a-CH bond of His-159 in the enzyme and the NH or 0 of the substrate-leaving group would destabilize the ES complex and so contribute to the observed kinetic specificity. Experimental support for this hypothesis was provided by the fact that when the ester or amide group of the substrate was replaced by a nitrile, the specificity of the enzyme was reflected in their binding constants [4]. This hypothesis further predicted that replacement of the ester or amide group in the substrate by an aldehyde should lead to a potent competitive inhibitor but replacement by a methyl ketone would not.Westerik and Wolfenden have shown that Nacyf-aminoacetaldehydes are indeed potent inhibitors of papain [6] and in this investigation N-acyl-aminopropanones have been shown to be poor inhibitors of the enzyme.Westerik and Wolfenden suggested that the potency of the N-acyl-aminoacetaldehydes was probably due to their ability to form a hemithioacetal with the active-site cysteine r...

Bone tools have a long archaeological history, and have recently been shown to retain use-traces distinctive of different perishable crafting practices. When examined in a controlled way, these diagnostic use-traces can serve as proxies for the crafted forms the bone tools were used to produce (e.g., baskets, leather goods, etc.). However, a number of methodological stumbling-blocks have hindered the sharing of bone tool use-wear results in a consistent standardized format. We suggest the application of Reflectance Transformation Imaging (RTI), and provide details for how to construct an RTI system which resolves most problems related to reproducibility in use-trace analysis.