Site-site heterogeneity in isocitrate lyase of castor seed endosperm: evidence from kinetics of inactivation by tetranitromethane

Inactivation of isocitrate lyase (native and EDTA-dialysed) by excess tetranitromethane (TNM) exhibits, biphasic kinetics, in which half of the initial activity is lost in a fast and the remaining half in a slow phase each following the pseudo-first order kinetics. Rate constants of the two phases are proportional to the TNM concentration. High succinate concentration protects the enzyme against TNM inactivation only in the slow phase without any effect on the fast phase. With the EDTA-dialysed enzyme, no such protection (against inactivation by TNM) is observed in the presence of succinate or Mg2+ ions. Addition of both these ligands together brings about protection against the slow phase (as with the native enzyme). It has been proposed that the site-site heterogeneity of isocitrate lyase is a consequence of its quaternary structure constraints.     

Kinetics of Chemical Modification of Arginine Residues in Mitochondrial Creatine Kinase from Bovine Heart: Evidence for Negative Cooperativity

The kinetics of chemical modification of arginine residues in mitochondrial creatine kinase (mit-CK) from beef heart by 4-hydroxy-3-nitrophenylglyoxal (HNPG) have been studied with simultaneous registration of enzyme inactivation. Experiments showed that complete inactivation of mit-CK corresponded to modification of two arginine residues per mit-CK monomer. The data on the modification kinetics can be described by the sum of two exponential terms and suggest strong negative cooperativity in the binding of HNPG to arginine residues. The rate constants for the fast and slow phases of modification differ by a factor of about 50. The corresponding rate constants for inactivation differ by a factor of about 30. The rate constant for the slow stage of inactivation is twice as large as that for the rate constant for the slow stage of modification, i.e., the inactivation process is ahead of the modification process.     

pH-dependent conformational transformation in mung bean glyceraldehyde-3-phosphate dehydrogenase

In thermal inactivation at pH 7.3 and below, the tetrameric apo-glyceraldehyde-3-phosphate dehydrogenase of mung beans lost half of its activity more rapidly than the rest, suggesting a pairwise arrangement of subunits (or a C2 symmetry). At pH 8.6, the activity was lost in a single exponential decay, characteristic of functional identity of sites as in a tetrahedral arrangement of subunits (or a D2-type symmetry). At intermittent pH values, the kinetics of thermal inactivation were consistent with the presence of a mixture of C2- and D2-symmetry conformations. In "sudden pH change" experiments, the observed thermal inactivation kinetics were characteristic of the final pH at which the enzyme was heated. Thus, the interconversion of the two conformations is facile and very fast. There is no gross change in molecular weight of the enzyme between pH 7.3 and 8.6.     

Essential arginine residues of creatine kinase from beef heart mitochondria

Creatine kinase from beef heart mitochondria is inactivated by 2,3-butanedione. The kinetics of inactivation of the mitochondrial enzyme is biphasic with a bend at a point corresponding to 50% inactivation. The inactivation rate constants of the first fast and the second slow phases of the reaction differ by one order of magnitude, thus suggesting the existence of two types of arginine residues, i.e. "fast" and "slow" ones, with different reactivities. The inactivation rate constant of the slow phase is very close to that for cytoplasmic creatine kinase. At saturating concentrations MgATP and MgADP afford complete protection of the slow phase of inactivation. It is assumed that the "slow" arginine is involved in the binding of metal-nucleotide substrates in the enzyme active center.     

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.     

Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water-in-oil microemulsion system

1. Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water-in-oil microemulsion, which consists of discrete nanometer-diameter water droplets dispersed in a continuous water-immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface. 2. Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule. 3. Studies are presented on the enzyme-catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations. 4. The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis-Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.     

Kinetic evidence for two Na channels in frog skeletal muscle

The kinetics of voltage-clamped sodium currents were studied in frog skeletal muscle. Sodium currents in frog skeletal muscle activate and inactivate following an initial delay in response to a depolarizing voltage pulse. Inactivation occurs via a double exponential decay exhibiting fast and slow components for virtually all depolarizing pulses used.The deactivation of Na currents exhibits two exponential components, one decaying rapidly, while the other decays slowly in time; the relative amplitude of the two components changes with the duration of the activating pulse. The two deactivation phases remain after pharmacological elimination of inactivation.In individual fibers, the percent amplitude of the slow inactivation component correlates with the percent amplitude of the slow deactivation component.Tetrodotoxin differentially blocks the slow deactivation component.These observations are interpreted as the activation, inactivation and deactivation of two subtypes (fast and slow) of Na channels.Studies of the slow deactivation phase magnitude vs the duration of the eliciting pulse provide a way to determine the kinetics of the slow Na channel in muscle.Ammonium substitution for Na in the Ringer produces a voltage dependent activation and inactivation of current which exhibits only one decay phase, and eliminates the slow decay phase of current, suggesting that adjustments of the ionic environment of the channels can mask the presence of one of the channel subtypes.     

猪心乳酸脱氢酶(H_4)在盐酸胍变性过程中失活与内源荧光变化速度的比较

本文比较了大然乳酸脱氢酶和硫酸铵稳定的乳酸脱氢酶在盐酸胍性过程式中失活与内源荧光的变化速度.酶失活表现为三相反应,即极快相,其速度常数用停流装置也无法测定;快相和慢相,1M胍变性时,此二相的一级反应速度常数分别为2.7×10~(-3)秒~(-1)和4.17×10~(-4)秒~(-1).在2M硫酸铵存在条件下,用2M胍更性时,快相和慢相的一极反应速度常数分别为6.16×10~(-3)秒~(-1)和1.88×10~(-3)秒~(-1).内源荧光强度的变化表现为二相反应,即极快相,相当酶失活的极快相,但变化幅度远小于酶失活的变化幅度;快相,相当于酶失活的快相,其速度常数为失活速度常数的1/3倍.上述结果表明,类似肌酸激酶,乳酸脱氢酶的失活速度快于酶分子整体构象的变化,相对于整个酶分子来说,活性中心的构象变化对变性剂更加敏感.     

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.     

Mechanism of reduction of Corynebacterium sarcosine oxidase by dithiothreitol

The mechanism of the reduction of Corynebacterium sarcosine oxidase [EC 1.5.3.1] by dithiothreitol (DTT) was investigated. The reduction followed biphasic kinetics with second-order rate constants of 54 M-1 X S-1 and 5.4 M-1 X S-1 for the respective phases. When the oxidized enzyme was titrated with sarcosine under anaerobic conditions, no intermediate, such as a semiquinone or a charge-transfer complex, appeared during the reduction of the enzyme. On the other hand, on DTT titration, an intermediate with a semiquinoid character appeared, and its formation was maximum when half of the total FAD was reduced. An oxidized semiapoenzyme, which had lost 45% of the noncovalently-bound FAD present in the native enzyme, also showed biphasic kinetics in the reduction with DTT. The second-order rate constant was found to be 38 M-1 X S-1 for the fast phase. An intermediate was also formed and its concentration, estimated by electron spin resonance (ESR) measurement, was found to agree with that of the noncovalently-bound FAD. In addition, the oxidized semiapoenzyme, which had lost 95% of the noncovalently-bound FAD present in the native enzyme, was reduced with DTT much more slowly than the native enzyme. In this case, the second-order rate constant was found to be 0.4 M-1 X S-1, and no intermediate was observed during the titration with DTT. On the basis of these data, it is suggested that the noncovalently-bound FAD accepts electrons directly from DTT in the fast phase through the semiquinoid form, while the covalently-bound FAD accepts electrons from the reduced noncovalently-bound FAD in the slow phase without forming an intermediate.     

Kinetics of jack bean urease inhibition by 2,3-dichloro-1,4-naphthoquinone. Elucidation of the mechanism: redox cycling and sulfhydryl arylation

The inhibition of jack bean urease by 2,3-dichloro-1,4-naphthoquinone (DCNQ) was studied at ambient temperature in 20?mM phosphate buffer, pH 7.8. The process was investigated by incubation procedure in the absence of substrate. It was found that DCNQ acted as a time- and concentration-dependent inactivator of urease. The time course of the reaction displayed a biphasic mode. Each phase followed a pseudo-first-order kinetics, however the inactivation rate at the first phase was significantly faster than at the next one. The biphasity indicated the complex mechanism of DCNQ action on urease. Quinones action on proteins has been elucidated as at least two processes: direct arylation of essential protein thiols and/or indirect oxidation of essential thiols by reactive oxygen species (ROS) realising during quinone reduction to semiquinones. The next evidence of the studied mechanism was provided by the reactivation experiment that showed the participation of reversible and irreversible processes in the inactivation. The application of dithiothreitol (DTT) into DCNQ blocked-urease solution resulted in an effective enzyme activity regain which quickly returned to 70?±?10%. The irreversible inactivation of urease was attributed to DCNQ arylation of thiol residues in the protein. On the other hand, it was assumed that the reversible inactivation was a result of the action of ROS such as H₂O₂. Presence of H₂O₂ in the incubation system was proved by an experiment with the use of catalase. The enzyme by the elimination of H₂O₂ decreased DCNQ inactivating influence on urease. The comparison of participation of the fast and slow phase in the inactivation with the percentage of the process reversibility was assumed that the fast period was a result of the arylation mechanism while the slow phase was related to the oxidative influence of H₂O₂.     

S-(2,3-dichlorotriazinyl)glutathione. A new affinity label for probing the structure and function of glutathione transferases.

S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 +/- 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme (K(d) 107.9 +/- 2.1 micro m for the fast phase, and 224.5 +/- 4.2 micro m for the slow phase) before irreversible modification with maximum rate constants of 0.049 +/- 0.002 min(-1) and 0.0153 +/- 0.001 min(-1) for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), approximately 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of alpha-helix H"'(3) and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites.     

High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems

The stability and structure of protein-containing water-in-oil (w/o) microemulsions were investigated by using the large protein immunoglobulin G (IgG, MW 155,000) in a mixture comprised of brine, sulfosuccinic acid bis [2-ethylhexyl]ester (sodium salt), and isooctane. We explored factors affecting the initial uptake of IgG into the w/o microemulsion and its subsequent release to a solid (precipitate) phase, and the kinetics of the latter process. Influences of such parameters as pH, ionic strength, and protein concentration on the solubilization and precipitation of bovine IgG in the organic phase are described. The structure and dynamics in microemulsions containing bovine IgG were probed by using dynamic light scattering, and it was found that the presence of IgG in the microemulsion induced strong attractive forces between the droplets. Based on results obtained by using these various experimental approaches, a model for protein solubilization and release is proposed. In this model, we propose the formation of clusters within which bovine IgG resides and which substantially slow the kinetics of protein release from the droplets to the precipitate phase.     

Study on the effect of some group-specific agents on clostridiopeptidase

The interaction of clostridiopeptidase of Clostridium histolyticum with EDC, TNM and MA, the specific reagents for COOH-groups, tyrosine and lysine residues was studied. It was shown that at pH 6.0 EDC inactivates the enzyme. The inactivation process follows the pseudo-first order kinetics and is described by a second order rate constant equal to 1 M-1 min-1. The synthetic substrate does not prevent, in practical terms, the enzyme inactivation by EDC. At pH 8.0 TNM modifies about 19 tyrosine residues in the clostridiopeptidase molecule which is accompanied by marked inhibition of the enzyme activity (down to 70-90%). In this case, the inactivation process is not described by simple pseudo-first order kinetics but is characterized by two steps (fast and slow) with second order rate constants of approximately 14 and 3.5 M-1 min-1, respectively. The synthetic substrate partly prevents the inactivation of the enzyme by TNM and protects 11 tyrosine residues. The MA-induced incorporation of 13 +/- 3 maleyl groups into the clostridiopeptidase molecule in partially prevented by the synthetic substrate with protects the enzyme against inactivation. The data obtained suggest that lysine residues are seemingly included into the active center of clostridiopeptidase, whereas tyrosine residues provide for the maintenance of active conformation of the enzyme.     

Esterification reactions catalyzed by Chromobacterium viscosum lipase in CTAB-based microemulsion systems

Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been used for multigram-scale ester synthesis, including the kinetic resolution of a secondary alcohol. The stability of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of ethylhexadecanoate as a model reaction. Under pseudo-first-order conditions, the synthesis rates were linearlydependent on the enzyme and fatty acid concentrations and the R dependence shows the characteristic bell-shaped curve (where R = [H(2)O]/[surfactant]). The dependence of enzyme activity toward octyldecanoate synthesis on the pH of the dispersed buffer phase is in marked contrast to that observed for the pH dependence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the former case, the pH-activity profile is approximately sigmoidal, which may reflect the ionization state of the fatty acid substrate. In the latter case, the pH dependence is minimal at both R = 10 and R = 50, suggesting the enzyme does not experience a changed pH environment. Inclusion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected buffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the esterification reactions, thereby providing an additional means of determining initial rates. The method may be of general value for lipase assays in microemulsions since it may provide, at the same time, information regarding enzyme regioselectivity. (c) 1995 John Wiley & Sons, Inc.     

Two components of voltage-dependent inactivation in Ca(v)1.2 channels revealed by its gating currents

Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Ca(v)1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V(1/2)) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge "immobilization," the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li(+) carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations.     

Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.     

Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron(III)-adriamycin complex.

The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

Non-equivalency of SH groups essential for the activity of mitochondrial creatine kinase

The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.