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Biomedical subjects

A M Klibanov

Publications and source records attributed to A M Klibanov.

At least 91 records · Page 5Linked to original sources
PubMed1982

Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography.

Alcohol Oxidoreductases↗
PubMed1981

The effect of electron carriers and other ligands on oxygen stability of clostridial hydrogenase.

The effects of various electron carriers, a substrate (H2) and a reversible inhibitor (CO) on the rate of irreversible oxygen inactivation of clostridial hydrogenase (ferredoxin: H+ oxidoreductase, EC 1.18.3.1) have been studied kinetically. Some electron carriers (e.g., clostridial ferredoxin and methyl viologen) greatly stabilize the enzyme, some (FAD, FMN) drastically reduce its stability, while others (benzyl viologen and methylene blue) only slightly alter the stability. Competitive experiments indicate that stabilizers and destabilizers do not compete with each other for binding with the active center of hydrogenase. Hydrogen and CO do not affect the rate of the oxygen inactivation. On the basis of the results obtained herein and kinetic data on hydrogenase catalysis from the literature, it is concluded that the active center of this hydrogenase comprises at least three different independent subsites. The first one (presumably an iron atom of the iron-sulfur cluster) binds H2 and CO and does not contribute to the oxygen stability. The second one binds stabilizers like methyl viologen while the third one binds destabilizers like FMN and FAD.

Benzyl Viologen↗
PubMed1979

Chelating agents protect hydrogenase against oxygen inactivation.

The effect of chelation on rate or air inactivation of hydrogenase from Clostridium pasteurianum has been investigated. All chelating agents used, whether water-soluble or water-insoluble, afforded protection against oxygen inactivation. EDTA appeared to be the most effective. Thus, in the absence of EDTA, hydrogenase in aqueous solution was nearly totally inactivated after 1 hour incubation in air, whereas 0.5 M EDTA (which did not affect significantly catalytic activity) allowed 41% retention of the initial activity even after 3 days incubation.

Chelating Agents↗
PubMed1979

The principles of enzyme stabilization. IV. Modification of 'key' functional groups in the tertiary structure of proteins.

The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.

Acetylation↗
PubMed1978

The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.

Adenosine Triphosphatases↗
PubMed1978

A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase.

A general procedure for stabilization of O2-labile enzymes exploiting "salting out" of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been developed. Empirical verification of this rationale is provided. The half-life of air inactivation of the O2-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20- to 25-fold simply by adsorption (noncovalent binding) in dilute Tris.HCl buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2. Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme. Most of the total catalytic activity, assayed as H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed.

Adsorption↗
PubMed1977

A new mechanochemical method of enzyme immobilization.

A new mechanochemical method for enzyme immobilization has been elaborated. The principle of this method consists of the following precepts. Partially hydrolyzed nylon fiber, the surface of which is known to be strewn with microcracks, is reversibly stretched (approximately 25%) and placed into an enzyme solution. Then, in the same solution, the fiber is made to relax and is taken out. The fiber retains considerable enzymatic activity even after numerous thorough washings (in a similar procedure without fiber stretching, equivalent washing removed all the enzymatic activity from the fiber). Immobilization on the fiber proceeds due to trapping of enzyme molecules by the microcavities on the surface of the support. The catalytic activity of mechanochemically immobilized chymotrypsin and trypsin is commensurable with their activity on covalent immobilization on nylon (calculated per unit of the macrosurface). A wide range of commercial polymers may be made of use as supports in the mechanochemical method of immobilization.

Chymotrypsin↗
PubMed1976

Enzymatic mechanochemistry: a new approach to studying the mechanism of enzyme action.

1. Covalent binding of model enzymes, chymotrypsin and trypsin, to elastic polymer supports, nylon and viscose (cellulose) fibers, human hair, methacrylate rubber, has been effectuated. On mechanical stretching of the fibers, the catalytic activity of the enzymes bound to them decreases, and when they relax, it increases to the initial level. The data obtained by us fit the concept that the effect is due to reversible deformation of the bound enzyme molecules induced by fiber stretching. 2. Analysis of the dependence of the catalytic activity of the enzymes chemically bound to the fiber on the degree of fiber deformation shows that the reversible inactivation of the enzymes induced by support stretching occurs even if the deformation of the enzymes' molecules is as small as 0.5 A. 3. The deformation of the enzyme molecules induced by fiber stretching entails a change in the substrate specificity of the biocatalysts, i.e. the activity towards "good" substrates decreases, and towards "poor" substrates increases. 4. The deformation of the enzyme molecules induced by fiber stretching results in a decrease of the specific catalytic activity of the biocatalyst, whereas its thermal stability increases. 5. The results obtained allowed a new, mechanochemical, approach to be suggested for studying major problems of enzymatic catalysis.

Binding Sites↗