Electrostatic interactions of 4-carboxy-2,6-dinitrophenyllysine-modified cytochromes c with physiological and non-physiological redox partners

An analysis of the effect of electrostatic properties of 4-carboxy-2,6-dinitrophenyllysine (CDNP-lysine) cytochromes c on their reactions with strongly and weakly binding redox partners is given. For strongly binding systems (cytochrome-c oxidase, cytochrome-c reductase, sulphite oxidase and yeast cytochrome-c peroxidase) the magnitude of the dipole moments of the CDNP cytochromes c determines their relative reactivities. For weakly binding redox agents, such as hexacyanoferrate(III), cobalt(III)tris(1,10-phenanthroline), azurin and plastocyanin, the electrostatic potential at the haem edge accounts for the greater part of the relative activities. Relative rate data were obtained from the literature. It is concluded that the dipole moment of native cytochromes c may account for an approx. 50-fold increase in the efficiency of its physiological activity towards membrane-bound enzymes. A correction on a formula to describe the contribution of a molecular dipole moment to the ionic strength dependence of a bimolecular rate constant (Koppenol, W. H. (1980) Biophys. J. 29, 493-508) leads to an equation nearly identical to that obtained by Van Leeuwen et al. (Van Leeuwen, J.W., Mofers, F.J.M. and Verrman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434-439).     

Photoinduced electron-transfer reaction in a ternary system involving zinc cytochrome c and plastocyanin. Interplay of monopolar and dipolar electrostatic interactions between metalloproteins.

A carbodiimide promotes noninvasive cross-linking between amino groups surrounding the exposed heme edge in zinc cytochrome c and carboxylic groups in the acidic patch in plastocyanin. Eight derivatives of the covalent complex Zncyt/pc(I), which have similar structures but different overall charges because of different numbers and locations of N-acylurea groups, are separated by cation-exchange chromatography. Kinetics of electron transfer from the diprotein complex in the triplet excited state, 3Zncyt/pc(I), to free cupriplastocyanin at pH 7.0 and various ionic strengths is studied by laser flash spectroscopy. This reaction is purely bimolecular for all eight N-acylurea derivatives of the diprotein complex. The overall charges of the derivatives 1 and 2 at pH 7.0 are -2 and 0, respectively; both of them, however, have very large dipole moments of 410-480 D. The rate constants for their reactions with cupriplastocyanin, whose charge at pH 7.0 is -8 and whose dipole moment is 362 D, are determined over the range of ionic strengths from 2.5 mM to 3.00 M. The observed dependence of the rate constants on ionic strength cannot be explained in terms of net charges (monopole-monopole interactions) alone, but it can be fitted quantitatively with a theory that recognizes also monopole-dipole and dipole-dipole interactions [van Leeuwen, J. W. (1983) Biochim. Biophys. Acta 743, 408]. At ionic strengths up to ca. 10 mM monopole-monopole interactions predominate and Br?nsted-Debye-Hückel theory applies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.     

The carboxyl modifier 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) inhibits half of the high-affinity Mn-binding site in photosystem II membrane fragments

The diphenylcarbazide(DPC)/Mn2+ assay [Hsu, B.-D., Lee, J.-Y., & Pan, R.-L. (1987) Biochim. Biophys. Acta 890, 89-96] was used to assess the amount of the high-affinity Mn-binding site in manganese-depleted photosystem II (PS II) membrane fragments from spinach and Scenedesmus obliquus. The assay mechanism at high DPC concentration was shown to involve noncompetitive inhibition of only half of the control level of DPC donation to PS II by micromolar concentrations of Mn at pH 6.5 (i.e., one of two DPC donation sites is inhibited). At low DPC concentration both DPC and Mn2+ donate to PS II additively. Treatment with the carboxyl amino acid modifier 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) inhibited half of the high-affinity Mn-binding site in spinach and Scenedesmus WT PS II membranes and all of the available site in Scenedesmus LF-1 mutant PS II membranes. A similar EDC concentration dependence was observed in all cases. Addition of 2 mM MnCl2 to the 10 mM EDC modification buffer provided complete protection for the Mn-binding site from modification. This protection was specific for Mn2+; six other divalent cations were ineffective. We conclude that EDC modifies that half of the high-affinity Mn-binding site that is insensitive to the histidine modifier diethyl pyrocarbonate (DEPC) [Seibert, M., Tamura, N., & Inoue, Y. (1989) Biochim. Biophys. Acta 974, 185-191] and directly affects ligands that bind Mn. The effects of EDC and DEPC that influence the high-affinity site are mutually exclusive and are specific to the lumenal side of the PS II membrane. Removal of the two more loosely bound of the four functional Mn from PS II membranes uncovers that part of the high-affinity site associated with carboxyl but not histidyl residues. We suggest that carboxyl residues on reaction center proteins are associated with half of the high-affinity Mn-binding site in PS II and are involved along with histidine residues in binding Mn functional in the O2-evolving process.     

Magnetic field-induced increase of the yield of (bacterio)chlorophyll emission of some photosynthetic bacteria and of Chlorella vulgaris.

In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emmission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.     

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].     

Phenylglyoxal modification of the Photosystem II reaction center

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance (ESR), have been used in conjunction with fluorescence-induction and dye-reduction assays to monitor electron transport in Photosystem II (PS II) subchloroplast particles incubated with the covalent modifier, phenylglyoxal. Phenylglyoxal-modified digitonin (D-10) particles from spinach are characterized by a high initial fluorescence yield (Fi) and an abolition of the variable component of fluorescence (Fv); an inhibition of PS-II-mediated reduction of dichlorophenol indophenol (DPIP) by sym-diphenylcarbazide; an abolition of flash-induced absorption transients (t1/2 greater than 2 microseconds) at 820 nm attributed to the primary electron donor, P-680+; the inhibition of photoreduction of the acceptor Qa; and the elimination of the ESR Signal 2s and Signal 2f. These observations suggest the critical participation of specific arginine residues on both the oxidizing and reducing sides of Photosystem II and also implicate phenylglyoxal as a quinone-binding site inhibitor (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271).     

Magnetic field-induced increase of the yield of (bacterio)chlorophyll emission of some photosynthetic bacteria and of Chlorella vulgaris

In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547–554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll.In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.     

Precursors to microsecond delayed luminescence in oxygenevolving and inhibited chloroplasts

We have investigated submillisecond delayed luminescence in spinach chloroplasts under a variety of conditions. In Tris-washed chloroplasts, which are inhibited on the oxidizing side of P-680, the delayed light emission in the 7–200 μs time-range decayed with biphasic behavior. In fully dark-adapted samples illuminated by a single saturating laser pulse, the fast phase of delayed luminescence followed a nearly identical pH-dependent time-course as that observed optically and by ESR for P⁺-680 reduction, thus verifying the recombination hypothesis for the origin of delayed light. The observed slower phase of delayed luminescence was also pH dependent, but unlike the fast phase, could not be ascribed to specific electron transfer events of PS II. This phase could be rationalized by a heterogeneity in the population of P-680. While kinetic parameters were found to be insensitive to changes in ionic strength, the overall luminescence intensity was quite sensitive to the electrical parameters, thus indicating the role of ionic strength and local charges in delayed luminescence modulation. A similar series of experiments was performed on untreated chloroplasts. The pH-dependent delayed luminescence behavior in both untreated chloroplasts and Tris-washed chloroplasts was similar despite significantly faster kinetics associated with the reduction of P⁺-680 by the secondary PS II electron donor, Z, in the former preparation (e.g., Van Best, J.A. and Mathis, P. (1978) Biochim. Biophys. Acta 503, 178–188). Thus, it was concluded that, in untreated samples, microsecond delayed luminescence emanates primarily from centers which are not competent in oxygen evolution. The nearly identical delayed luminescence intensity in untreated chloroplasts and in Tris-washed chloroplasts was rationalized by a model which predicts modulations in delayed luminescence yield by the exciton-quenching effect of P⁺-680. Computer simulations demonstrate the feasibility of this model. The previously documented flash oscillations in microsecond delayed luminescence intensity in untreated chloroplasts (Bowes, J.M. and Crofts, A.R. (1979) Biochim. Biophys. Acta 547, 336–346), which we readily observed, were attributed to alterations in delayed luminescence yield (in nonfunctional centers) by variations in charge density stored at the oxygen-evolving complex of functional centers. Taken together, our results emphasize the dependence of delayed luminescence kinetics upon electron-transfer kinetics and the dependence of delayed luminescence amplitude upon the photochemical parameters, the exciton yield and the emission yield.     

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin.

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.     

Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones

Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins. Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp. azurin, and Alcaligenes sp. nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times). Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin. The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin. This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved. The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude). This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.     

On the mechanism of channel-length dependence of gramicidin single-channel conductance

Single-channel conductance data on four different gramicidin channel lengths demonstrate that conductance magnitude is neither inversely dependent on the square of the channel length nor on the image force arising from differences in the extent of lipid dimpling (Jordan and Vayl (1985) Biochim. Biophys. Acta 818, 416-420). Rather the conductance differences are consistent with the decreased off-rate constant for the singly occupied state as the ionic radius decreases from that of cesium ion to sodium ion coupled with the decreased probability of the doubly occupied channel due to increased ion-ion repulsion as the channel is shortened (Urry et al. (1984) Biochim. Biophys. Acta 774, 115-119).     

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.     

Do copper ions influence the reduction of ferricytochrome C by O-2?

Recently, it was suggested that the measured rate of reduction of ferricytochrome C by O-2 below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157-168). The high values were attributed to the presence of impurities of copper, which compete for O-2. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome C in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome C. It was previously shown by us and by others that the reduction yield of ferricytochrome C by O-2 IS 100%. We measured the rate of reduction of ferricytochrome C by Cu(I), and found that this reaction is slow: k = (1.5 +/- 0.5) . 10(3) M-1 . s-1. Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome C by O-2.     

Structure of the copper sites in membrane-bound cytochrome c oxidase

The structures of membrane proteins are difficult to obtain by crystallography and may be altered by the detergents used in their extraction. X-ray absorption spectroscopy has been used to identify the structures of the copper atoms of the membrane-bound enzyme in mitochondria and in submitochondrial particles at respective concentrations of 100 and 200 micron of molar copper. To within the experimental error, the x-ray absorption spectra of the copper atoms of the membrane-bound and the Yonetani (Yonetani, T. (1961) J. Biol. Chem. 236, 1680-1688) purified oxidase are identical; all detectable shells of the active site indicate no alteration of structural parameters. Significant differences are found when compared to the Hartzell-Beinert (Hartzell, R. C., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) preparation. Extended x-ray absorption fine structure technology is now adequate for the direct studies of membrane proteins in situ in their natural environment.     

Post-translational modification of human erythrocyte pyruvate kinase

Upon storage, partially purified human erythrocyte pyruvate kinase (ATP: pyruvate-phosphotransferase, E.C. 2.7.1.40) from normal individuals was found to undergo a spontaneous oxidation to a form which displayed markedly reduced activity. This modified form of the enzyme exhibited kinetic patterns similar to those frequently reported for the enzyme in cases of nonspherocytic hemolytic anemia. The data are discussed in relation to the recently proposed theory that post-translational modification of pyruvate kinase is responsible for the abnormal kinetic patterns frequently encountered for this enzyme in the disease state. [Van Berkel, T. J. C., Koster, J. F., Kruyt, J. K. and Staal, G. E. J. 1973 $\text{Biochim. Biophys. Acta 321}$, 496–502].     

The N-terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin-stimulated peripheral blood human lymphocyte receptors

Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).     

Hydrogen-deuterium exchange studies on guanidinated pig heart lactate dehydrogenase

Pig heart lactate dehydrogenase becomes more thermostable on increasing the degree of guanidination (conversion of lysine to homoarginine) (Minotani, N., Sekiguchi, T., Bautista, J.G. and Nosoh, Y. (1979) Biochim. Biophys. Acta 581, 334-341). The conformational change of the protein on guanidination was then examined by hydrogen-deuterium (H-2H) exchange reactions. It ws found that (i) the fluctuation degrees of peptides and tyrosine and tryptophan residues in the protein decrease in that order, (ii) two H-2H exchangeable tryptophan residues per subunit are freely accessible to solvent and the fluctuation degrees of the residues does not change on guanidination, (iii) the H-2H exchange detectable tyrosine residues are not freely accessible to solvent and become less fluctuating when 15 lysine residues per subunit are guanidinated, and (iv) the peptides become much less fluctuating on increasing the degree of guanidination. The specific activity of the enzyme decreased on guanidination. The increased thermostability of the protein on guanidination may be related to the decrease in flexibility of the molecular structure by sacrificing the enzyme activity.