Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol.61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.     

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.     

Inactivation and conformational changes in methyl parathion hydrolase in 2,2,2-trifluoroethanol solutions: Inactivation kinetics and molecular dynamics simulation

Many improvements have been made in the understanding of functional and structural characteristics of proteins in a denaturant-based microenvironment. This study reports the chemical denaturation of methyl parathion hydrolase (MPH, EC 3.1.8.1) using 2,2,2-trifluoroethanol (TFE). MPH is an important enzyme that catalyzes the hydrolysis of organophosphorus agents. However, the regulation of MPH activity and structural changes during unfolding are not well studied, particularly for TFE unfolding. We investigated MPH unfolding with TFE for the first time. In this study, changes in enzymatic activity and unfolding of MPH at different TFE concentrations were investigated by enzyme activity measurements, intrinsic fluorescence and by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectral scans. The results showed TFE inactivated MPH in a dose-dependent manner. A Lineweaver–Burk plot analysis revealed that the type of inhibition was reversible noncompetitive inhibition. Intrinsic fluorescence and ANS-binding fluorescence showed that TFE induced obvious tertiary structural changes in MPH by exposing hydrophobic groups. Furthermore, we conducted a docking simulation between MPH and TFE. The computer simulation successfully showed the binding structure and we estimated stability by calculating the binding energy (lowest binding energy: -3.18 kcal/mol). The results demonstrate that MPH can be inactivated by TFE, and provide new insights into the mechanism of TFE-induced unfolding of MPH and inhibition of ligand binding.     

Protein conformational changes induced by guanidine at predenaturational concentrations

The nature of the molecular event that apomyoglobin undergoes at predenaturational concentrations of guanidine has been investigated by means of steady-state and multifrequency phase and modulation fluorometry. The results have been compared to those observed for liver alcohol dehydrogenase. From these studies has been hypothesized a different susceptibility of the distinct elements of secondary, super-secondary, and tertiary structure towards the denaturing action of guanidine at predenaturational concentrations.     

Inactivation of creatine kinase by high pressure may precede dimer dissociation.

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.     

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.     

Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe

A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.     

Interaction of brain-type creatine kinase with its transition state analog: kinetics of inhibition and conformational changes

The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The Km for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and the Ki was 0.16 mM; in the presence of the transition state analog, MgADP + NO3- + creatine, the Ki was estimated to be 0.04 mM. Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel filtration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 +/- 0.05 M-1 min-1 to 6.96 +/- 0.11 M-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration-induced conformational and flexibility changes in lysozyme at low water content

Using laser Raman spectroscopy, we are able to study conformational changes that occur as previously-dried hen egg-white lysozyme is sequentially rehydrated. Parallel n.m.r. exchangeability studies enable us to monitor flexibility changes also during this rehdyration. The results are consistent with a general loosening up of the protein at a water content of ～0.08 g water/g protein, followed by (probably small) local conformational changes. The enzyme regains its activity only after both these processes have gone to completion; thus these solvent-related changes may be necessary before activity can recommence.     

Studies on the conformational changes of metalloproteins induced by electrons in water-ethylene glycol solutions at low temperatures. III. Adrenal ferredoxin.

The reduction of adrenal ferredoxin (adrenodoxin) at low temperatures was investigated in order to separate local modifications of the active centre of the protein on its reduction, from the conformational transition which seems to accompany the change of the redox state of the irons; The ESR spectra of the states of the protein, where the reduced active centre is to be found by the "oxidized" conformation of the apoprotein, were obtained. The transition from the states of the protein to the state which occurs on its chemical reduction at room temperature was also investigated. The results of the work support the view that conformational changes in proteins (enzymes) which take place while they are functioning proceed after modifications of the active centres (change of the redox state, adsorption of a substrate, etc.), and are essentially caused by them. Adrenal ferredoxin was the third subject in our studies of the intermediate states of proteins which appear after reduction of their active centres by means of electrons trapped in water-ethylene glycol mixtures at the temperature of liquid nitrogen [1, 2]. In the reduced state, the active centre of the protein has an ESR signal with a g-factor of 1.94 [3, 4] which is convenient for our purposes.     

Inactivation of poliovirus and other enteroviruses by solutions of sodium fluoride at low pH

Solutions of sodium fluoride at pH 3 to 4 inactivated enteroviruses, whereas other sodium salts had little or no effect on virus infectivity. Solutions of potassium fluoride also inactivated viruses under similar conditions. Light, temperature, and the presence of organic compounds such as detergents and fecal matter did not affect inactivation of virus by 0.4 M solutions of sodium fluoride at pH 3. to 4. Decreasing the sodium fluoride concentration below 0.04 M or raising the pH above 4 reduced the viricidal properties of the solutions. Virus adsorbed to membrane filters and sludge flocs could not be recovered after treatment of solids-associated virus with solutions of sodium fluoride.     

Protein conformational changes and myelin solubilization by anion-detergent solutions.

The addition of sodium sulfate to a myelin suspension in sodium phosphate buffer at neutral pH, containing octyl glucoside detergent (OG), increases the membrane solubility more than 5-fold by an unknown structural mechanism. FTIR spectroscopy has been applied to investigate anion effects on the conformational structure of myelin proteins. Sulfate and sulfate-phosphate media, but not phosphate alone, induce a great conformational protein disorder. The addition of the detergent to the anion mixture solution prevents the myelin from protein denaturation. The conformational transitions have also been quantified through the amide I region. Explanations of these changes and their connections with myelin solubility are also included.     

Inactivation of creatine kinase induced by dopa and dopamine in the presence of ferrylmyoglobin

We investigated the effect of dopa and dopamine on creatine kinase (CK) activity in the presence of ferrylmyoglobin (ferrylMb). CK was sharply inhibited by dopa and dopamine in the presence of ferrylMb. Dopa and dopamine markedly promoted the reduction of ferrylMb to metmyoglobin (metMb). The semiquinone from dopa and dopamine may be involved in CK inactivation. During inactivation of the enzyme, both kinetic parameters Vmax and Km changed. In addition, reduced glutathione restored the activity of CK at an early stage. These results suggest that inactivation of CK is dominantly due to oxidation of sulfhydryl (SH) groups of the enzyme. Other catechols, such as adrenaline and noradrenaline, little inactivated CK activity, whereas they promoted the reduction of ferrylMb to metMb. Other SH enzymes, including alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were inactivated to a lesser extent by dopa and dopamine in the presence of ferrylMb. Adrenaline and noradrenaline did not significantly prevent the inactivation of ADH and very slightly inhibited GAPDH. These results suggest that dopa and dopamine act as prooxidants to inactivate SH enzymes in the presence of ferrylMb.     

Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268

Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.