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Biomedical subjects

A L Fink

Publications and source records attributed to A L Fink.

At least 109 records · Page 6Linked to original sources
PubMed1987

Kinetic and structural characterization of reversibly inactivated beta-lactamase.

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.

Bacillus cereus↗
PubMed1987

Characterization of the unfolding of ribonuclease A in aqueous methanol solvents.

The effect of methanol on the thermal denaturation of ribonuclease A has been investigated over the -40 to 70 degrees C range. The transition was fully reversible to at least 60% (v/v) methanol at an apparent pH of cryosolvent (pH) of 3.0 and was examined at methanol concentrations as high as 80%. The unfolding transition, as monitored by absorbance change at 286 nm, became progressively broader and occurred at increasingly lower temperatures as the alcohol concentration increased. In 50% methanol, increasing the pH from 2 to 6 shifted the transition to higher temperature. A substantial decrease in cooperativity was noted at the more acidic conditions. On the other hand, increasing concentrations of guanidine hydrochloride in 50% methanol caused the transition to shift to lower temperatures with little effect on the cooperativity. The observed effects on the cooperativity of the unfolding transition suggest that methanol and lower temperatures may increase the concentration of partially folded intermediate states in the unfolding of ribonuclease. Comparison of the transition in 50% methanol as determined by absorbance or fluorescence, which monitor the degree of exposure of buried tyrosines and hence the tertiary structure, to that determined by far-UV circular dichroism, which monitors secondary structure, indicated that the major unfolding transition occurred at a higher temperature in the latter case. Thus, the tertiary structure is lost at a lower temperature than the secondary structure. This observation is consistent with a model of protein folding in which initially formed regions of secondary structure pack together, predominantly by hydrophobic interactions, to give the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-Ion Concentration↗
PubMed1987

Cryoenzymology of human plasmin catalysis: comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates.

A comparative study of nucleophilic (methanol), aprotic (dimethyl sulfoxide), and protic but non nucleophilic (ethylene glycol, ethylene glycol/dimethylformamide) solvents on the catalytic and structural properties of human plasmin has been made. All four solvent systems are potentially suitable as cryosolvents for plasmin catalysis at subzero temperatures although the solubility of plasmin is limited in the methanol and dimethyl sulfoxide systems. Each cryosolvent system caused minor effects on the catalytic properties of the enzyme, which could be rationalized in terms of the known physical properties of the cosolvent. Solvent systems containing ethylene glycol induce a minor conformational change which increases the catalytic efficiency of plasmin. The cosolvent effects on Km and Ki indicate that electrostatic interactions dominate the binding of both substrates and inhibitors such as benzamidine. A change in slope of the Arrhenius plots for catalysis, reflecting a temperature-induced isomerization, is observed around 0 degree C; the energies of activation being 13 +/- 2 kcal mol-1 at higher temperatures and 19 +/- 2 kcal mol-1 at subzero temperatures, and essentially independent of solvent. Deacylation was shown to be the rate-limiting step in the hydrolysis of specific p-nitrophenyl ester substrates. Previous stopped-flow studies at room temperature provided observations suggesting that a tetrahedral intermediate could be detected in the plasmin-catalyzed hydrolysis of p-nitroanilide substrates. Experiments at subzero temperatures with such substrates failed to reveal any buildup of a tetrahedral intermediate under the experimental conditions.

Anilides↗
PubMed1986

Effects of methanol cryosolvents on the structural and catalytic properties of bovine trypsin.

The effect of aqueous methanol cryosolvents on the catalytic and structural properties of bovine trypsin has been investigated. The low freezing points and low viscosities of methanol-based cryosolvents are desirable for a variety of cryoenzymological experiments. Increasing concentrations of methanol caused increases in the values of kcat and Km for the hydrolysis of N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at 0 degrees C and a small increase in Ki for inhibition by benzamidine. Based on product analysis the increase in kcat with increasing methanol concentration at pH* 4.0 and 6.5 can be completely accounted for by nucleophilic competition of methanol for the acyl enzyme intermediate. This observation indicates that deacylation is the rate-limiting step under these conditions. The effect of increasing methanol concentration on kcat/Km for the above ester substrate and N alpha-benzoyl-L-arginine p-nitroanilide was similar. Incubation experiments indicated that trypsin was quite stable in 70% methanol at 0 degrees C and below. The Arrhenius plot for the catalytic reaction in 70% methanol was linear over the 0 to -40 degrees C range, indicating no change in rate-determining step nor temperature-induced structural perturbation. No evidence for structural effects induced by methanol or temperature were detected by monitoring the intrinsic fluorescence and absorbance. We conclude that aqueous methanol cryosolvents are satisfactory for cryosolvent studies of trypsin.

Dimethyl Sulfoxide↗
PubMed1986

Effects of cryoprotectants on enzyme structure.

The interaction between organic cosolvents and proteins is considered, especially from the point of view of effects on protein stability. It is concluded that each protein-cosolvent system constitutes a unique situation, making generalized predictions of expected effects difficult. Two classes of cosolvents are distinguished, based on the nature of their interactions with the protein surface. The thermodynamic instability to the system introduced by the presence of the cosolvent can be accommodated (i) by preferential exclusion of the cosolvent from the vicinity of the protein, (ii) by major structural changes of the protein, or (iii) by aggregation. Polyols tend to undergo preferential exclusion due to unfavorable interactions with nonpolar surface groups, whereas monohydric alcohols and other more hydrophobic cosolvents may undergo preferential exclusion due to adverse interactions with charged groups on the protein surface. Typical cosolvent effects on the structural and catalytic properties of enzymes are illustrated with data for ribonuclease and beta-lactamase with alcohol cosolvents. The relative hydrophobicity of the cosolvent is the major determinant of the effect of a cryosolvent on the enzyme stability and properties. Thus the position of the unfolding transition in cryosolvent will be decreased more by a more nonpolar cosolvent. Different cosolvents can have significantly different effects on the catalytic and structural properties of the same enzyme. Conversely the same cosolvent can have significantly different effects on similar proteins. The number and distribution of the nonpolar and charged groups on the protein's surface probably are the major determinants of the protein contribution to the solvent-protein interaction. The large temperature dependence of the rates of protein unfolding and refolding can be beneficially utilized in cryoprotectant studies of living cells.

Alcohols↗
PubMed1984

Cryoenzymology of porcine pepsin.

Physical and kinetic properties of porcine pepsin have been examined in aqueous methanol solvents at temperatures below ambient to seek evidence for covalent intermediates in the catalyzed hydrolysis of good substrates. It was first demonstrated that aqueous methanol cryosolvents have no significant deleterious effects upon this protein. The addition of methanol does lead to a drastic reduction in the midpoint of the thermal melting curve of pepsin. This could account for rate reductions previously observed in catalysis by this enzyme. This effect is lessened by the addition of active-site ligands including substrates and is fully reversible upon dilution into aqueous solution. Two substrates were chosen which have chromophoric groups on opposite sides of the scissile peptide bond. The UV spectral changes from hydrolysis of Pro-Thr-Glu-Phe-(NO2)Phe-Arg-Leu and the fluorescence spectral changes from hydrolysis of DNS-Ala-Ala-Phe-Phe-OP4P+-CH3 were studied at temperatures down to -60 degrees C. The resulting Arrhenius plots were linear in the region where pepsin exists in the native state with downward curvature exhibited at higher temperatures where the reversible denaturation occurs. No "burst" reactions were observed with either substrate. In addition, efforts at trapping intermediates by low-temperature denaturation and precipitation have provided no evidence for covalent intermediates on the reaction pathway. Although this evidence is negative, we cannot rule out the possibility of the formation of covalent intermediates following an initial rate-limiting step.

Animals↗
PubMed1984

Cryoenzymology of penicillopepsin; with an appendix: mechanism of action of aspartyl proteinases.

Intrinsic spectral and kinetic properties of penicillopepsin and its action on N-acetylalanylalanyllysyl-p-nitrophenylalanylalanylalanine amide have been investigated at subzero temperatures in aqueous methanol and dimethyl sulfoxide solutions in an attempt to find evidence for or against a covalent mechanism in the catalyzed hydrolysis of peptide bonds. The study of fluorescence and circular dichroism spectra as a function of solvent concentrations gave no evidence for any solvent-induced structural effects at temperatures below the thermal denaturation transition. The effect of temperature on the intrinsic fluorescence of penicillopepsin in either 60% (v/v) methanol or 50% (v/v) dimethyl sulfoxide did not indicate any temperature-induced structural changes. On the other hand, Arrhenius plots for the hydrolysis reaction over the range 0 to -50 degrees C showed downward curvature. A probable explanation for this phenomenon is that the reduction in flexibility of the enzyme due to thermal and viscosity factors leads to the stabilization of a nonproductive conformation. The pH optima of kcat/Km are shifted from 5.1 in aqueous solvents to 5.6 in 60% methanol and to 6.6 in 50% dimethyl sulfoxide. Aqueous methanol caused small decreases of Km and of Kcat; the decrease in the latter was greater than that brought about by the decrease in the water concentration. In aqueous dimethyl sulfoxide, there was no detectable change in kcat up to 15%, but Km increased by more than an order of magnitude. Above 15%, only kcat/Km could be measured. No evidence for the accumulation of either covalent amino or covalent acyl intermediates was obtained when penicillopepsin was incubated at -70 degrees C in 67% methanol with several substrates. Although negative, these experiments do not rule out conclusively the involvement of covalent intermediates in penicillopepsin-catalyzed reactions.

Aspartic Acid Endopeptidases↗
PubMed1984

Low-temperature reactions of trypsin with p-nitroanilide substrates: tetrahedral intermediate formation or enzyme isomerization.

The reactions of trypsin with the p-nitroanilides of N alpha-carbobenzoxy-L-lysine, N alpha-carbobenzoxy-L-arginine, and N alpha-benzoyl-L-arginine have been studied in the 0 to -30 degrees C temperature region, over a range of pH values, using 65% (v/v) aqueous dimethyl sulfoxide cryosolvent. At alkaline pH, -30 degrees C, the catalytic reaction appears as a slow "burst" of product (or intermediate) followed by turnover. For all three substrates, the rate of the burst phase is identical. Preincubation of the enzyme at -30 degrees C abolishes the burst. On addition of trypsin to the cryosolvent at -30 degrees C, a time-dependent decrease in fluorescence emission is observed with the same rate as that of the burst with the anilides. The burst phase is thus interpreted as reflecting a temperature/solvent-induced isomerization of trypsin to a less catalytically efficient form, rather than the previously suggested formation of a tetrahedral intermediate [Compton, P. D., & Fink, A. L. (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. The isomerization is not observed at high temperature (greater than or equal to 0 degree C) or at neutral pH. The burst phase was not observed with aqueous methanol cryosolvent, indicating that it is sensitive to both cosolvent and temperature.

Arginine↗
PubMed1983

The effect of cryosolvents on the spectral and catalytic properties of liver alcohol dehydrogenase.

The effects of aqueous organic cryosolvents on the structural and catalytic properties of horse liver alcohol dehydrogenase (liver alcohol dehydrogenase) have been investigated. The cosolvents studied were ethanol, methanol, dimethyl sulfoxide, and dimethylformamide. All show potential as cosolvents for cryoenzymological investigations of the catalytic action of liver alcohol dehydrogenase. Limitations due to the formation of abortive complexes, or the cosolvent acting as a substrate were considered. Possible adverse structural effects of the cosolvents were ascertained by utilizing the intrinsic fluorescent properties of the enzyme. Catalytic effects, as inferred from steady state kinetic studies, were determined from both the oxidation of ethanol and the reduction of p-nitroso-N,N-dimethylaniline, a chromophoric aldehyde analog. It is concluded that each of these solvent systems may be useful for studying certain aspects of the liver alcohol dehydrogenase catalytic mechanism at subzero temperatures. Thus, although the formation of ternary enzyme-cosolvent complexes may restrict the use of cosolvents in some experiments, no apparent adverse effects are observed on the enzyme structure, coenzyme binding, or catalytic reactions. A number of interesting features were observed. For example, fluorescence titration of the native enzyme near 0 degrees C in either aqueous solution or 50% dimethyl sulfoxide revealed pK values in the vicinity of 10.5 and 12.5, in contrast to the previously reported single pK of 9.2 observed in aqueous solution at 25 degrees C.

Alcohol Dehydrogenase↗
PubMed1982

Observation of intermediates in the folding of ribonuclease A at low temperature using proton nuclear magnetic resonance.

The refolding of ribonuclease A (RNase A) has been investigated in aqueous methanol cryosolvents in the 0 to -20 degrees C range. When a thermally unfolded sample was brought under renaturing conditions (e.g., -16 degrees C, 35% methanol, pH 2.8), the refolding, as monitored by the absorbance change at 286 nm (which reflects the degree of solvent exposure of Tyr), was triphasic and took approximately 1 h for completion. The 360-MHz proton nuclear magnetic resonance (NMR) spectrum of the native enzyme in either 35% or 50% aqueous methanol is very similar to that in aqueous solution. When the refolding of RNase A was monitored in the subzero temperature range with the signals of the His C2 protons, new resonances rapidly appeared, in addition to those from native protein. The new resonances are attributed to a partially folded intermediate state that has a relatively compact structure. Time-dependent changes were observed in the areas of the resonances from both native and partially folded species. The rates of peak area reduction for the intermediate state were the same as those for the increase in area of the native resonances, and similar to those for the second phase observed in the absorbance experiments. The results are consistent with the slow-refolding form of RNase A consisting of a least two distinct populations. A model for the folding of RNase A is proposed.

Endoribonucleases↗
PubMed1981

Cryoenzymology.

A detailed understanding of the mechanism of enzyme action requires a correspondingly detailed knowledge of the structures of the intermediates and transition states on the catalytic pathway, as well as the kinetics and thermodynamics of their interconversion. Cryoenzymology, the use of subzero temperatures and fluid cryosolvents, has the potential to supply this type of information. In this article recent investigations illustrating the advantages of cryoenzymology are reviewed. The major advantage lies in the ability to accumulate and stabilize productive enzyme-substrate intermediates for sufficient lengths of time to allow the collection of high resolution structural data, e.g. by X-ray diffraction. In addition, intermediates which are not detectable under normal conditions, due to too short lifetimes and/or low concentrations, may be detected at low temperatures, in some cases through changes in the rate-limiting step. The method seems of rather general applicability, judging by the number of different types of enzymes, including oligomeric and membrane-associated ones, which have successfully been studied using cryoenzymology. In some cases, at least, good agreement between observations at subzero temperatures and normal conditions has been found, demonstrating the relevance of the technique. Potential limitations of the techniques, as well as questions regarding the effects on the protein structure, are also discussed.

Animals↗
PubMed1980

Temperature-dependent change in the rate-limiting step of beta-glucosidase catalysis.

When beta-glucosidase from sweet almonds reacts with p-nitrophenyl-beta-D-glucoside at subzero temperatures, a rapid release of p-nitrophenol, stoichiometric with enzyme concentration, occurs prior to turnover. This observation is interpreted as reflecting rapid formation of a glucose-enzyme intermediate, followed by rate-limiting breakdown. The energies of activation for the formation and breakdown of the intermediate were 6.0 +/- 2 and 19.0 +/- 2 kcal mol-1, respectively. Extrapolation of the reaction rates to higher temperatures suggests an isokinetic temperature circa 24 degrees C. Measurement of kcat in aqueous solution as a function of temperature yields a breakpoint in the Arrhenius plot at 20 degrees C and activation energies above and below this point which correspond to the values at subzero temperatures (6.7 and 17.0 kcal mol-1, respectively). This breakpoint thus reflects a change in the rate-determining step from formation of the intermediate at higher temperatures to breakdown at lower values, and explains why a "burst" of p-nitrophenol is observed at low, but not high, temperatures with this substrate. The significance of this finding to other glycohydrolases, and enzyme catalysis in general, is considered.

Glucosidases↗