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Biomedical subjects

A M Klibanov

Publications and source records attributed to A M Klibanov.

At least 73 records · Page 4Linked to original sources
PubMed1988

Why is one Bacillus alpha-amylase more resistant against irreversible thermoinactivation than another?

Half-lives of Bacillus alpha-amylases at 90 degrees C and pH 6.5 greatly increase in the series from Bacillus amyloliquefaciens to Bacillus stearothermophilus to Bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. This stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of B. amyloliquefaciens and B. stearothermophilus alpha-amylases, so that for B. licheniformis alpha-amylase, another process, deamidation of Asn/Gln residues, emerges as the cause of inactivation. The extra thermostability of the thermophilic enzyme was found to be mainly due to additional salt bridges involving a few specific lysine residues (Lys-385 and Lys-88 and/or Lys-253). These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures, consequently making it less prone to forming incorrect (scrambled) structures and thus decreasing the overall rate of irreversible thermoinactivation. The implications of these findings for protein engineering are discussed.

Amides↗
PubMed1988

Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases.

Molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile Bacillus amyloliquefaciens and from the thermophile Bacillus stearothermophilus, have been elucidated in the pH range of relevance to enzymatic catalysis. At pH 5.0, 6.5, and 8.0, B. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. At the last pH, this process can be suppressed by the presence of the substrate starch and consequently a covalent process, deamidation of asparagine and/or glutamine residues, becomes the cause of loss of enzymatic activity at 90 degrees C. Monomolecular conformational scrambling is the predominant cause of irreversible inactivation of B. stearothermophilus alpha-amylase at 90 degrees C at pH 5.0, 6.5, and 8.0. At pH 6.5 another contributing inactivation mechanism is the deamidation of amide residues, and at pH 8.0, O2 oxidation of the enzyme's cysteine residue.

Acetamides↗
PubMed1988

Enzymatic catalysis in nonaqueous solvents.

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.

Catalysis↗
PubMed1987

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme.

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.

Amino Acid Sequence↗
PubMed1987

Thermal destruction processes in proteins involving cystine residues.

To ascertain the upper limit of protein thermostability and to evaluate the introduction of additional -S-S- bridges for the enhancement of protein thermostability by site-directed mutagenesis, the stability of cystine residues at 100 degrees C has been investigated in a number of unrelated proteins. All proteins studied (more than a dozen) undergo heat-induced beta-elimination of cystine residues in the pH range from 4 to 8. The time courses of this process closely follow first-order kinetics indicating that the stability of a cystine residue is not significantly affected by its position in the polypeptide chain. Furthermore, the first-order rate constants of beta-elimination of disulfide bonds at 100 degrees C are remarkably similar for different proteins--0.8 +/- 0.3 h-1 and 0.06 +/- 0.02 h-1 at pH 8.0 and 6.0, respectively. Thus, this process is relatively independent of both the primary structure and the elements of higher structures remaining in proteins in 100 degrees C. beta-Elimination of disulfides produces free thiols that cause yet another deleterious reaction in proteins--heat-induced disulfide interchange. This reshuffling of -S-S-bonds, which is much faster than beta-elimination, has also been quantitatively characterized.

Cystine↗
PubMed1987

Enzyme-catalyzed, gas-phase reactions.

Dehydrated preparations of alcohol oxidase adsorbed on DEAE-cellulose vigorously catalyze a gas-phase oxidation of ethanol vapors with molecular oxygen. The gas-phase reaction is strongly dependent on the water activity of the system. The enzymatic activity is severely inhibited by the product hydrogen peroxide. This inhibition can be alleviated, however, by an addition of catalase or peroxidase to the dry preparation. Such dehydrated, bienzymic catalysts afford a complete and selective conversion of the substrate to acetaldehyde. Dry alcohol oxidase is much more thermostable than in aqueous solution. The results of this work suggest that dehydrated enzymes have potential applications in the analysis of gaseous compounds and in the development of novel gas-solid bioreactors.

Alcohol Oxidoreductases↗
PubMed1987

Control of oligomeric enzyme thermostability by protein engineering.

The ability to control the resistance of an enzyme to inactivation due to exposure to elevated temperatures is essential for the understanding of thermophilic behavior and for developing rational approaches to enzyme stabilization. By means of site-directed mutagenesis, point mutations have been engineered in the dimeric enzyme yeast triosephosphate isomerase that improve its thermostability. Cumulative replacement of asparagine residues at the subunit interface by residues resistant to heat-induced deterioration and approximating the geometry of asparagine (Asn-14----Thr-14 and Asn-78----Ile-78) nearly doubled the half-life of the enzyme at 100 degrees C, pH 6. Moreover, in an attempt to model the deleterious effects of deamidation, we show that replacement of interfacial Asn-78 by an aspartic acid residue increases the rate constant of irreversible thermal inactivation, drastically decreases the reversible transition temperature, and reduces the stability against dilution-induced dissociation.

Carbohydrate Epimerases↗
PubMed1986

Why does ribonuclease irreversibly inactivate at high temperatures?

The mechanism of irreversible thermoinactivation of bovine pancreatic ribonuclease A in the pH range relevant to enzymatic catalysis has been elucidated. At 90 degrees C and pH 4, the enzyme inactivation is caused by hydrolysis of peptide bonds at aspartic acid residues (the main process) and deamidation of asparagine and/or glutamine residues. At 90 degrees C and neutral pH (pH 6 and 8), the enzyme inactivation is caused by a combination of disulfide interchange (the main process), beta-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. These four processes appear to demarcate the upper limit of thermostability of enzymes.

Animals↗
PubMed1986

Horseradish peroxidase catalyzed hydroxylations: mechanistic studies.

The hydroxylation of phenol to hydroquinone and catechol in the presence of dihydroxyfumaric acid and oxygen catalyzed by horseradish peroxidase was studied under conditions where the product yield was high and the side reactions were minimal. The reaction is partially uncoupled with a molar ratio of dihydroxyfumaric acid consumed to hydroxylated products of 12:1. Hydrogen peroxide does not participate in the reaction as evidenced by the lack of effect of catalase and by the direct addition of hydrogen peroxide. Conversely, superoxide and hydroxyl radicals are involved as their scavengers are potent inhibitors. Experiments were all consistent with the involvement of compound III (oxygenated ferrous complex) of peroxidase in the reaction. Compound III is stable in the presence of phenol alone but decomposes rapidly in the presence of both phenol and dihydroxyfumaric acid with the concomitant formation of product. Therefore, phenol and dihydroxyfumaric acid must be present with compound III in order for the hydroxylation reaction to occur. A mechanism consistent with the experimental results is proposed.

Animals↗
PubMed1986

Peroxidases depolymerize lignin in organic media but not in water.

Horseradish peroxidase and milk lactoperoxidase, while unable to degrade either synthetic or natural lignins in aqueous solutions, vigorously depolymerize polyconiferyl alcohol, milled wood lignin, and kraft pine lignin in dioxane, dimethylformamide, or methyl formate containing 5% aqueous buffer (10 mM acetate, pH 5). Horseradish peroxidase, solubilized in organic media by chemical modification, can also degrade lignin in native lignocellulose (wheat straw).

Journal Article↗
PubMed1985

Enzymatic removal of bilirubin from blood: a potential treatment for neonatal jaundice.

Current treatments for severe jaundice can result in major complications. Neonatal jaundice is caused by excessive accumulation of bilirubin in the blood. A small blood filter containing immobilized bilirubin oxidase was developed to reduce serum bilirubin concentrations. When human or rat blood was passed through the enzyme filter, more than 90 percent of the bilirubin was degraded in a single pass. This procedure may have important applications in the clinical treatment of neonatal jaundice.

Animals↗
PubMed1985

The mechanisms of irreversible enzyme inactivation at 100C.

The mechanism of irreversible thermoinactivation of an enzyme has been quantitatively elucidated in the pH range relevant to enzymatic catalysis. The processes causing irreversible inactivation of hen egg-white lysozyme at 100 degrees C are deamidation of asparagine residues, hydrolysis of peptide bonds at aspartic acid residues., destruction of disulfide bonds, and formation of incorrect (scrambled) structures; their relative contributions depend of the pH.

Animals↗
PubMed1985

Enzyme-catalyzed processes in organic solvents.

Three different lipases (porcine pancreatic, yeast, and mold) can vigorously act as catalysts in a number of nearly anhydrous organic solvents. Various transesterification reactions catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The dependence of the catalytic activity of the enzyme in organic media on the pH of the aqueous solution from which it was recovered is bell-shaped, with the maximum coinciding with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases can catalyze several other processes in organic media including esterification, aminolysis, acyl exchange, thiotransesterification, and oximolysis; some of these reactions proceed to an appreciable extent only in nonaqueous solvents.

Alcohols↗
PubMed1984

Enzymatic catalysis in organic media at 100 degrees C.

Porcine pancreatic lipase catalyzes the transesterification reaction between tributyrin and various primary and secondary alcohols in a 99 percent organic medium. Upon further dehydration, the enzyme becomes extremely thermostable. Not only can the dry lipase withstand heating at 100 degrees C for many hours, but it exhibits a high catalytic activity at that temperature. Reduction in water content also alters the substrate specificity of the lipase: in contrast to its wet counterpart, the dry enzyme does not react with bulky tertiary alcohols.

Alcohols↗
PubMed1983

Remarkable positional (regio)specificity of xanthine oxidase and some dehydrogenases in the reactions with substituted benzaldehydes.

Upon an increase in the size of the substituent, the reactivity of xanthine oxidase to ortho-substituted benzaldehydes drastically decreases while that to para-substituted benzaldehydes does not change significantly. The enzyme exhibits this regiospecificity with respect to both electron-withdrawing substituents (e.g., halogens) and electron-donating ones (alkyls and alkoxyls). Xanthine oxidase-catalyzed oxidation of m- and p-nitrobenzaldehyde is more than 300-times faster than that of the o-isomer, whereas the rates of their non-enzymatic oxidation are comparable, as are the rates of the enzymatic oxidation of p- and o-nitrocinnamaldehyde. These and other findings of this work indicate that the discovered positional specificity of xanthine oxidase is due to steric hindrances in the reaction of the enzyme's active center with the aldehyde moiety having a bulky substituent in its close proximity. Such regiospecificity of the enzyme exists regardless of the nature of the electron acceptor used and can be employed for the separation of mixtures of positional isomers of substituted benzaldehydes. A marked positional specificity in the xanthine oxidase-catalyzed oxidation of substituted benzaldehydes appears to be a rather general phenomenon: three other enzymes tested, alcohol dehydrogenases from horse liver and yeast and aldehyde dehydrogenase from yeast, all follow a similar pattern in the reactions with para- and ortho-substituted halobenzaldehydes.

Alcohol Dehydrogenase↗