Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Mechanistic studies of a tyrosine-dependent catalytic antibody.

A pre-steady-state multiple-turnover kinetic burst is observed during hydrolysis of phenyl acetate by the catalytic antibody, 20G9. The burst is caused by partial product inhibition by phenol (Ki,app = 2.5 microM), which lowers both kcat and KM by almost an order of magnitude without affecting kcat/KM. The acid limb of the steady-state kcat pH profile of native 20G9 has a pKa of 9.6, suggesting a catalytic role for tyrosine. Additional evidence for an essential tyrosyl residue is that mild treatment of 20G9 with tetranitromethane nitrates a single tyrosine per equivalent of antigen binding sites and the mononitrated derivative has less than 5% of the native activity. Near-UV absorbance spectroscopy suggests that the alternative substrates N-carbobenzoxyglycine O-phenyl ester (ZG-OPh) and N-acetylglycine O-phenyl ester (AcG-OPh) acylate multiple tyrosines on the antibody. Neither ZG-OPh nor AcG-OPh are measurably catalyzed once appreciable acylation has taken place. Antibody acylated by ZG-OPh is inactive toward phenyl acetate hydrolysis, but can be reactivated by hydroxylamine. The data and derived kinetic rate equations are consistent with an acyl mechanism for phenyl acetate hydrolysis in which phenol inhibits by binding to a covalent O-acetyltyrosyl intermediate, slowing deacylation. Although the data are consistent with such a mechanism, they do not rule out other plausible, yet less unifying mechanisms of phenol inhibition; the observed burst could conceivably result from partial mixed phenol inhibition or from phenol-induced nonproductive substrate binding. Because antibodies often use tyrosines in antigen binding, tyrosyl catalytic antibodies may be commonly encountered in the future.

Potentiometric biosensor employing catalytic antibodies as the molecular recognition element.

Catalytic antibodies are introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the catalytic reaction of the substrate. Consequently, molecular recognition by catalytic antibodies can yield reversible immunoblosensors. In this example, a prototype potentiometric biosensor is described in which a micro-pH electrode is modified with a catalytic antibody that catalyzes the hydrolysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response is linear with the log of substrate concentration over a range of 20-500 microM with a detection limit of 5 microM under the conditions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.

Structure of 6-(iodomethyl)-2-oxo-2-phenoxy-1,2-oxaphosphorinane.

C11H14IO3P, Mr = 352.11, monoclinic, P2(1)/n, a = 8.300 (3), b = 14.081 (2), c = 11.326 (4) A, beta = 104.32 (3) degrees, V = 1282.6 (13) A3, Z = 4, D chi = 1.823 Mg m-3, lambda(Mo K alpha) = 0.71073 A, mu = 25.8 cm-1, F(000) = 688, R = 0.035 for 2364 observed reflections. The six-membered ring O(1), P(2), C(3), C(4), C(5), C(6) is in a chair conformation with phenoxy and iodomethyl groups adopting axial and equatorial orientations, respectively. The bond distances and angles are unexceptional.

Catalysis of a stereospecific bimolecular amide synthesis by an antibody.

We report a nonhydrolytic bimolecular aminolysis reaction catalyzed by an antibody. The stereospecific formation of an amide from racemic lactone plus an aromatic amine is described by a random equilibrium bireactant kinetic sequence. The observed turnover rate may be approximated from the measured difference between the binding of reactants and the transition state analog.

A stereospecific cyclization catalyzed by an antibody.

A monoclonal antibody elicited by a transition-state analog that is representative of an intramolecular six-membered ring cyclization reaction acted as a stereospecific, enzyme-like catalyst for the appropriate substrate. Formation of a single enantiomer of a delta-lactone from the corresponding racemic delta-hydroxyester was accelerated by the antibody by about a factor of 170, which permitted isolation of the lactone in an enantiomeric excess of about 94 percent. This finding demonstrates the feasibility of catalytic-antibody generation for chemical transformations that require stereochemical control.