John A. Thoma

Alkaline protease associated with the matrix protein of a virus infecting the cabbage looper.

An alkaline protease was found to be tightly associated with the matrix protein of a nuclear-polyhedrosis virus infecting Trichoplusia ni . Partial purification of the protease was achieved by CaCl 2 precipitation of the matrix protein. The protease had a pH optimum of 9.5 using casein as substrate and behaved as a serine protease. It hydrolyzed N-benzoyl- l -tyrosine ethyl ester, and it was inhibited by Hg 2+ and Cu 2+ . Most of the protease was separated from the matrix protein by polyacrylamide gel electrophoresis at pH 9.5.

A possible mechanism for amylase catalysis

Abstract Arguments are presented which indicate that functional group catalysis by itself cannot account for the remarkable efficiency of amylases. On the basis of activation thermodynamics and X-ray analysis of lysozyme, a ring distortion is suggested as a possible additional interaction mechanism. To explain the exceptional rate of amylase catalysis, a reaction mechanism involving a combination of general acid catalysis by imidazole, ring distortion, and electrostatic solvation or general base catalysis or covalent bond formation with a carboxyl group is proposed. The overall stereochemistry of the reaction is ultimately controlled by the enzyme-directed approach of the solvent to the reaction center. The mechanism is consistent with both enzymic and physical organic data related to the hydrolysis of glycosides.

Degradation of matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni by an endogenous protease

Abstract The intact matrix protein of a nuclear-polyhedrosis virus of Trichoplusia ni could be isolated when a contaminating endogenous protease was inhibited by Hg 2+ . The subunit molecular weight of the intact matrix protein was approximately 28,000. When the protease was not inhibited, the matrix protein was degraded to a mixture of peptides which had a strong tendency to self-associate. The protease-degraded matrix protein preparation could be dissociated by 1.0 M formic acid into a single peptide and a peptide aggregate. The sum of the amino acid compositions of the peptide and peptide aggregate was identical, within experimental error, with the amino acid composition of the intact matrix protein.

Models for deploymerizing enzymes: criteria for discrimination of models

Several models for the action of alpha amylase have been proposed to account for the nonrandom distribution of oligosaccharides in the amylase digests of polysaccharides. The preferred-attack model attempts to account for the nonrandom distribution by assuming that the probability for bond cleavage depends upon the position of the bond in the chain. The repetitive, or multiple-attack, model suggests that the nonrandom distribution of oligosaccharides arises because an amylase can form a cage-like complex with a substrate and attack it several times during a single encounter. The multiple-enzyme or dual-site model suggests that the nonrandom yield of oligosaccharides arises from the combined action of exo- and endo-enzymes. The effects of pH, inhibitors, and substrate chain-length on enzyme action have been studied in several laboratories to determine which of the three action-patterns best describes the action of alpha amylase. The influence of these variables on product distributions or enzyme action-patterns are mathematically modeled in the Appendix. The experimental data on porcine-pancreatic alpha amylase are discussed in the light of the derivations.

Subsite mapping of enzymes: Collecting and processing experimental data—a case study of an amylase-malto-oligosaccharide system

Two research groups have independently developed the theory and experimental methodology for quantitatively assessing substrate monomer-subsite binding-energies for depolymerases. When the two approaches are applied to the same enzyme-substrate system they yield surprisingly divergent results. This paper outlines the application of the two approaches to an amylase-maltooligosaccharide system and points out the more important areas of disagreement. We show that by proper data-management, the conflicts between the tow laboratories are basically resolved. The complexities of the subsite model demand extensive data-gathering and exacting data-processing and verification that the computed model-parameters can faithfully reproduce the experimental data.

Subsite mapping of enzymes. Use of subsite map to simulate complete time course of hydrolysis of a polymeric substrate.

Abstract In this paper we extend our earlier work on subsite mapping and show that our model for depolymerase action can be used to accurately predict product ratios vs the extent of reaction when a polymer is hydrolyzed. The experimental product ratios for Bacillus amyloliquefaciens α-amylase acting on reducing end-labeled 14C-maltodextrins ranging in chain length 3 to 10 are reported. These data and Michaelis parameters are used with a depolymerase computer model (J. D. Allen, 1977, Ph.D. thesis, University of Arkansas; J. D. Allen and J. A. Thoma, 1976, Biochem. J.159, 105 ) to compute an optimized subsite map. The depolymerase computer model generates a 10-subsite map for B. amyloliquefaciens α-amylase with the catalytic site located to the left of subsite 7. The binding affinities of the subsites are then used as the sole input in another computer program to quantitatively predict the mole fraction of products vs the extent of hydrolysis for substrates of varying chain length. Excellent agreement is obtained between the computed and experimental data for seven maltodextrins examined.

Multiple forms of porcine pancreatic α-amylase

Abstract Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V max and K m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.

Repetitive attack by Aspergillus oryzae alpha amylase

The action of Aspergillus oryzae alpha amylase on reducing-end, and uniformly radiolabeled maltotriose through maltodecaose has been studied. The enzyme is found to hydrolyze more than a single glycosidic bond during enzyme-substrate encounters. The extent of this repetitive attack is quantitated.

Separation of factors responsible for lysozyme catalysis.

Abstract The four major factors contributing to lysozyme facilitation of acetal hydrolysis are general acid catalysis, distortion, electrostatic stabilization of the transition state and entropy effects. The importance of general acid catalysis is assessed from the Bronsted relationship using an a coefficient of 0·5 based on model compounds. The degree of electrostatic stabilization is assessed from the electric field interactions of Asp 52 and Glu 35. Interaction of lysozyme with transition state analogs is used to calculate the contribution of distortion to catalysis. The remaining acceleration factor is assigned to entropy effects. The value of the entropy effect suggests that the transition state has some freedom of motion on the enzyme.

Stable lysozyme-cello-oligosaccharide complexes

Abstract Stable complexes that formed when [ 14 C]cello-oligosaccharides and lysozyme were incubated under various conditions were isolated chromatographically and characterized. Complexation occurred over the pH range of 3–9 but was favored at high pH; the extent of complexation was inversely proportional to temperature. The stoichiometry of the complex was 1:1 sugar-protein, but no radiolabeled peptides could be isolated from a tryptic digest. The minimum requirements for association were native enzyme and a substrate composed of three or more (1→4)-β- D -glucopyranosyl residues. Acceptors and competitive inhibitors of lysozyme inhibited complexation and stimulated dissociation of the complex. Lysozyme did not hydrolyze the cello-oligosaccharides nor did it use them as D -glucosyl donors. Ultracentrifugation, molecular-sieve chromatography, and light-scattering studies indicated that the precipitated complexes were large, heterogeneous aggregates of protein and oligosaccharide which conform to the following equilibrium: n (protein+oligosaccharide)⇌(protein-oligosaccharide) n . Polymerization is a cooperative phenomenon.