摘要:
12 12 J.C.S. DaltonInteraction of Cysteine with Vitamin B12a : Kinetic and ThermodynamicInvestigationsBy Faruk Nome and Janos H. Fendler," Department of Chemistry, Texas A & M University, College Station,Texas 77843, U.S.A.Rate and equilibrium constants for the interaction of L-cysteine with vitamin B12* have been determined in aqueoussolutions in the absence and in the presence of air as functions of pH, buffer concentration, and temperature.Kinetic treatment of the data has afforded the pH-independent rate constants for the anation of aquo- and hydroxo-cobalamins by L-cysteine, for the aquation of the vitamin B,,-~-cysteine complexes, and for the formation ofvitamin BIzr from the L-cysteine complex of vitamin BlSa. The mechanism of these reactions is discussed.IN spite of their obvious importance, relatively fewdetailed kinetic studies have been carried out on ligand-exchange reactions with vitamin B1za.14 Rate constants1 J.M. Pratt, ' Inorganic Chemistry of Vitainin BIZ,' Aca-for the anation, k,, and those for the aquation, k+ ofvitamin Bib, and aquocobalamin (bzm-Co-OH,) havebeen determined for the ligands (L) N3-, OCN-, SCN-,R. H. Prince and D. A. Stotter, J. Inorg. Nuclear Chem.,G. N. Schrauzer, Pure Appl. Chem., 1973, 33, 646.demic Press, New York, 1972. 1973, 35, 321.D. G. Brown, Progr. In.org. Chem., 1973, 18, 1771976 1213SO,,-, NCO-, I-, Br-, imidazole, and glycine.&llAlthough the structure of cobalamin is far from simple,kibzm-Co-OH, + L +- bzm-Co-L + H,O (1)k-1chelation of the benzimidazole (bzm) side chain precludessubstitution in a stoicheiometry other than 1 : 1, at leastin the range pH 4-10.l Kinetic treatment of the datafor simple ligand substitutions of aquocobalamin is,therefore, unexpectedly simple.The recognized role of sulphydryl groups in reactionscatalyzed by vitamin B,, enzymes prompted the numer-ous exploratory investigations of the interactions ofthiols with methylcobalamin,12 aqu~cobalamin,~~-l~ andB,, coenzymes.l7 Surprisingly, however, there is onlymeagre information on the detailed kinetics of theseprocesses.Reaction of thiols with methylcobalamin wassuggested to result in the formation of vitamin B1a andthe methylated thiol with a subsequent one-electronreduction to yield vitamin B12s.3J2 Alternatively,vitamin Bizs was postulated to form directly in theinteraction of thiols with rnethyl~obalamin.*~~~-~~ Nosuch dispute exists, however, on the steps involved inthe interaction of thiols with vitamin Investig-ations of the kinetics of reaction (2) are, therefore, notwater; the pH of the buffered solutions was adjusted withNa[OH] and HC1 and was determined by means of a Radio-meter pHM-26 instrument.The pH of the solutions forkinetic runs a t temperatures other than 25.0 "C were cor-rected by the temperature compensator of the pH meter.Spectrophotometric determinations were made using aCary 118-C spectrophotometer whose cell compartment wasthermostatted a t 25.0 f 0.1 "C. Initially, the completespectral range was recorded, generally on the scale 0-2.0 Aat a speed of 10 nm in-1 and 0.2 nm s-l.Kinetic data wereobtained on a Cary 118-C spectrometer. Temperatures forthe kinetic runs were maintained by water circulation.Some kinetic runs were carried out in degassed solutions.For the slower runs, using the Cary 118-C spectrophoto-meter, solutions were placed in the two limbs of a speciallyconstructed vessel which additionally had a quartz cell anda high-vacuum stopcock attached to it. The solutions inthe separate compartments were degassed on a high-vacuumline by repeated freeze-pump-thaw cycles. Subsequent tothe removal of air (ca. 10-8 Torr), the high-vacuum stopcockwas closed and the vessel was removed from the vacuumline. The solutions were brought to the desired tempera-ture, rapidly mixed, and transferred to the limb containingthe quartz cell.Rate constants for the reactions of L-cysteine with thevitamin BIZ-cysteine adduct were determined in somesystems by pH jump.An unbuffered solution of vitaminH20 SRVitamin B ,zabzmVitamin B12only inherently important but they may provide addi-tional insight into the analogous methyl transfer. Thepresent work reports the results of our kinetic and ther-modynamic studies of L-cysteine with vitamin Bl% inaqueous solutions.EXPERIMEKTALThe best available grades of vitamin Blsa, aquocobalamin,bznrCo-OH, (Merck) , L-methionine (Sigma), L-cysteine(Sigma), and 2-mercaptoacetic acid and 3-mercaptopropionicacid (Aldrich) were used as received.Stock solutions of thethiols were prepared under a stream of nitrogen immediatelyprior to their use. Using these precautions, only a minimumof oxidation took place as established by the criteria ofreproducibility of rate measurements and of ultravioletabsorbaiice of the thiols for the duration of the experiments.All stock solutions were prepared in double glass-distilledW. C. Randall and R. A. Alberty, Biochemistry, 1966, 5,3189.6 W. C. Handall and R. A. Alberty, Biochemistry, 1967, 6,1620.D. Thiisius, Chem. Comm., 1969, 1183.J. G. Heathcote and M. A. Slifkin, Biochem. Biophys. Acta,0 J . G. Heathcote, G. H. Moxon, and &I. A. Slifkin, Spectro-lo D. Thusius, J . Amer. Chem. Soc., 1971, 93, 2629.l1 J. H. Fendler, F.Nome, and H. C. Van Woert, J . Amev.l2 G. -1gnes. H. A. 0. Hill. 1. M. Pratt. S. C. Ridsdale. I;. S.1968,158, 167.chim. Acta, 1971, A27, 1391.Chem. SOL, 1974, 96, 6745.Kennedy,-md I<. J. P. Williaks, Biochim'. Biophys. Acta,' 1971,252, 307.Blsa and L-cysteine a t ca. pH 4.5 was allowed to react tocompletion in one of the arms of the special degassing vessel,while the other arm contained a buffered solution adjustedto an appropriate, but at least three units higher, pH value.Subsequent to degassing, the two solutions were mixed andformation of vitamin BIzr was followed a t the appropriatewavelength.Polarographic titration of vitamin Blza with L-cysteinewas carried out by means of a Sargent model XXI polaro-graph. All reactions were followed under pseudo-first-orderconditions to at least 98% completion.Plots of log(A, -A,) against time were linear and the error in the reportedrate constants is f3%.RESULTSAddition of excess of L-cysteine, 2-mercaptoacetic acid,or 3-mercaptopropionic acid to a buffered (pH 5.5) solution13 N. Adler, T. Medwick, and T. J. Poznanski, J . Amer. Chem.SOC., 1966, 88, 6018.14 J. M. Pratt, J . Chem. SOC., 1959, 6164.16 H. A. 0. Hill, J. M. Pratt, R. G. Thorp, B. Ward, and R. J. P.Williams, Biochem. J., 1970,120, 263.16 G. N. Schrauzer and J. W. Sibert, Arch. Biuchewz. Biuphys.,1969,130, 257.17 P. Y . Law and J. M. Wood, J . Amev. Chem. SOL, 1973, 95,914.18 G. N. Schrauzer and R. J. Windgassen, Nature, 1967, 214,492.19 G. N.Schrauzer and R. J. Windgassen, J . Awzev. Chem. SOC.1967, 89, 3607.20 G. N. Schrauzer, Bioinorg. Chem., 1974,3, 3531214 J.C.S. Daltonof vitamin B12s resulted in marked alteration of the absorp-tion spectra (Table 1). Conversely, addition of excess ofmethionine or L-cysteine did not alter the spectra of aquo-cobalamin over a period of several days. The spectra of theproducts formed in the interaction of the three thiols withvitamin B128 were identical (Figure l), indicating the1I350 500 550 6k-A / nrnFIGURE 1 Absorption spectra of vitamin B,,-cysteine (a),vitamin Bla-3-mercaptopropionic acid (b), and vitamin B,-2-mercaptoacetic acid (c) adducts. Substrate concentrationswere 4.0 x 10-6 mol dm-3. Spectra were recorded in a 1.00 cmcell; those due to (b) and (a) are displaced by 0.1 and 0.2absorbance unitsformation of a product which is independent of the structureof the thiols.An identical spectra was also reported for theinteraction of glutathione with aqu0coba1amin.l~ Thecysteine did not change the spectra. This fact and theobserved isosbestic points at 337, 362, 445, and 632 nmindicate the equilibrium formation of a product betweenvitamin BlZa and L-cysteine. Addition of L-cysteine tovitamin Blze a t pH > 7.0 resulted in a somewhat morecomplex behaviour. Subsequent to a time lag of severalminutes, there was a time-dependent change in the ' limit-ing ' spectra of the vitamin B12,-~-cysteine adduct. Thetime lag was somewhat irreproducible and represents theconsumption of oxygen in the stoppered cell.In degassedbuffered solutions of L-cysteine and vitamin Blza a t pH>7.30, these changes were observed without the time lag.A typical time-dependent spectral change for this process isillustrated in Figure 2 ( b ) . Compared to the limiting spectraof the vitamin B12,-~-cysteine adduct [Figure 2(a)], absorb-ances increased a t 400 and 470 nm a t the expense of thoseat 370, 530, and 652 nm with isosbestic points a t 388 and 491nm. The limiting spectrum is stable for several days andcorresponds to that attributed to vitamin I312r.l Theobserved spectral changes are compatible with the reactionsequence :bzm-Co-OH, + RSH -- bzm-Co-SHR + H20klaPP*(3)k-laPP*k ,RPP*bzm-Co-SHR + RSH __+ Vitamin Bl2r + RS- (4)Table 1 gives absorption maxima and absorption co-efficients for aquocobalamin, hydroxocobalamin, the vitaminB12-thiol complexes, and vitamin B12r.Absorption co-efficients were related to those of vitamin Blea and conse-quently they are independent of the amounts of water ofcrystallization in our samples. Development of the vitaminB,,-L-cysteine adduct and vitamin Blzr could be followedTABLE 1Absorption spectral parameters in water a t 25.0 "CAm.,. 10-4 & Amax 10-4 & &ax. 10-1 E - - -Species nm dma mol-l cm-l nm dm3 mol-l cni-1 nm dm3 mol-1 cm-lbzm-Co-OH, 360 2.60 497 0.79 523 0.83(360) a (2.62) abzm-Co-OH 357 2.10bzm-Co-L-cys teine 370 1.40 632 0.75 ti62 0.74(370) (1.41)bz1n-Co-3-mercaptopropionic acid 371 1.40 634 0.83 564 0.79bzm-Co-2-mercaptoacetic acid 371 1.38 634 0.76 562 0.74Vitamin B,,5 From ref, 1.stoicheiometry of the interaction of cysteine with aquo-cobalamin was established by polarography.Titration ofa 1.0 x mol dm-3 solution of vitamin B12a with a con-centrated solution of L-cysteine in the polarography cell,using 0.1 mol dm-3 sodium acetate at pH 5.5 or 0.1 mol dm-3sodium tetraborate a t pH 9.3 as supporting electrolytes,gave end-points a t 8.5 x 10-5, 8.0 x 9.5 x and8.7 x mol dm-3 cysteine. Interaction of thiols withvitamin Blza is, therefore, likely to involve formation of a1 : 1 complex.Development of the absorption spectra of the vitaminB1--thiol adducts was dependent on the concentration ofthe thiols. This is illustrated for L-cysteine in Figure 2(a).Gradual addition of L-cysteine to vitamin B12r, a t pH < 7.0resulted in increases in the absorbances a t 552 and 370 nmat the expense of the absorbances a t 495 and 525 nm.A tsufficiently high cysteine concentration, addition of further40 1 0.72 471 0.93(402) a (0.76) a (473) a (0.92)b From ref. 12.spectrophotometrically. Knowledge of the appropriatespectral parameters (Table 1) allows meaningful dissectionof KlaPP. and k2app. [equations (3) and (4)J. Rate constantsfor the equilibrium attainment of the vitamin BlZa--~-cysteine complex [equation (3)] were best obtained in air-saturated solutions a t wavelengths where bzm-Co-SHR andvitamin Blzr have isosbestic points (388 and 490 nm).Similarly, the formation of vitamin B12r from bzm-Co-SHR [equation (4)] could be best followed a t wavelengthswhere bzm-Co-OH2 and bzm-Co-SHR have isosbesticpoints (337, 362, 445, and 532 nm).The observed pseudo-first-order rate constants, ,%$, for the interaction of L-cys-teine with vitamin Blw a t different pH values, buffer con-centrations, and temperatures are given in Table 2. Theerror in the second-order rate constants is considered to be&8%. Using the method described in the Experimentalsection, reactions (3) and (4) were independently determine1976 1215for the interaction of L-cysteine with vitamin B12a at the of these plots yielded rate constants for the formation of theappropriate isosbestic points (see above). Data obtained vitamin B12,-~-cysteine complex, i.e.values for RlaPP-.for these separate processes are also given in Table 2. Values of klaPP- at a given pH were independent of theThe observed pseudo-first-order rate constants for the concentration of buffers (Table 3). Furthermore, theequilibrium attainment of vitamin BlS-L-cysteine complex reaction proceeded at the same rate in the dark as in the3 60 LOO LLO 4 80 520 560/nmFIGURE 2 Absorption spectra of (a) 5.0 x 10-5 mol dm-3 vitamin Blzs a t pH 5.6 in water (- - - ),inthepresenceof3.6 x6.0 xIn01 dw3) in water a t pH 7.3 and 2 (- - - -), 12 (- - - -), 30 (-), 80 (. * .), and 1SO min (-) after mixing(- - - - -), and 1.0 x mol dm-3 mol dm-3 L-cysteine (-), (b) of the vitamin B,,-L-cysteine adduct (6.0 xformation, h4, allows, under favourable conditions, the light.Anation of bzm-Co-OH,, therefore, is not photo-calculation of the apparent (i.e. pH-dependent) rate constant catalyzed. Since for most cases the intercepts of these linesfor the anation, klaPP., and aquation, h-laPP., from equation did not diverge appreciably from the origin, values of k-laPP*(5). Good linear relations were obtained for all systems must be small and cannot be meaningfully obtained bytreatment of the kinetic data accordingto equation (5).Attempts were made to obtain the equilibrium constantson plotting the data according to equation (5). Gradients for the -formation of vitamin Blga--L-cysteine adducts12 16 J.C.S. DaltonCondi-tions bADAAdA "AADAdA "AADAAAAAA dA "DADDDDAAAAAATABLE 2Interaction of L-cysteine with vitamin Blza a103[~-Cysteine] lmol dm-3A0.50 0.67 0.83 1.00 1.50 2.00 2.33 2.50 2.66102k$h/s-' A - nm pH , A3 503503503503503503503503503503503503503523543553573573573573573573.854.324.45L O O c 1.23 1.52 1.895.OOc 2.94 3.08 4.205.075.205.505.50e 1.26 1.51 1.875.50" 2.94 3.03 4.285.786.4016.50 f7.20f7.50 f8.17 f8.689.079.308 0.396 0.480 0.6059.30g 1.03 1.19 1.579.409.40362, 7.30532362, 8.85532362, 9.20532362, 9.65532350 5.07350 5.07 3352 7.20"352 7.201357 9.07'"357 9.07n1.701.511.652.20 3.355.37 8.881.771.951.932.10 3.455.25 8.601.701.751.411.201.400.900.660.750.764 1.151.98 3.010.770.612.221.982.101.992.312.342.252.222.101.701.721.190.931.060.820.862.622.432.272.752.952.572.562.402.061.341.141.121.031.050.026 0.0300.137 0.197l .l O h 1.50h2.24h 2.90h1.63 2.13 2.571.74 2.30 2.401.22 1.68 2.381.21 1.73 2.120.77 1.08 1.100.76 1.05 1.103.122.722.582.832.882.80 3.513.202.882.312.401.611.351.381.181.150.0290.2241.753.422.882.702.462.411.401.403.00 3.33 3.5b\3.503.213.283.563.503.203.843.102.562.661.651.601.531.301.194.303.483.733.984.124.122.953.232.131.691.781.131.440.0320.2891.98 A4.023.12 3.853.20 3.822.56 2.912.70 2.951.55 1.761.50 1.753.464.013.300.0350.3302.314.65In water at 25.0 OC, unless stated otherwise.[Vitamin B12,] = 5.0 x3 0.50 mol dm-3 Sodium acetate buffer.mol dm-3. Followed by use of the Cary 118 instru-ment. A = Air saturated, D = degassed. c 0.10 mol dm-3 Sodium acetate buffer. At 35.0 OC. At 45.0 "C. f 0.10 mol dm-3Sodium dihydrogenphosphate buffer. Average values from two wavelengths.i 0.30 mol dm-3 Sodium acetate buffer. " 0.30 mol dm-3 Sodium dihydrogonphosphatebuffer. 0.075 in01 dm-3Sodium tetraborate buffer.g 0.10 mol dm-3 Sodium tetraborate buffer.0.50 mol dm-3 Sodium dihydrogenphosphate buffer. m 0.050 mol dmrP Sodium tetraborate buffer.TABLE 3Apparent kinetic and thermodynamic parameters for the reaction of L-cysteine with vitamin BIza in aqueous solutions3.85 A4.00 A4.32 D4.45 A5.001 A5.00g A5.07 A5.07e A5.07 A5.20 A5.50 A5.50 D5.50f A6.50g A5.78 A6.40 A7.20 A12.110.810.522.867.012.012.211.911.511.522.663.011.513.2 5.43 21.69.755.41 21.76.06 d 23.27.2OC A 9.857.20" A 9.607.30 D 0.0417.50 A 9.508.17 A 6.808.40 A 5.96d 8.388.68 A 6.308.85 D 0.9409.07 A 5.079.07 A 5.159.07 t A 4.959.20 D G.109.30f A 10.09.309 A 26.09.40 A 4.259.40 D 4.359.65 D 12.1a In water a t 25 "C and 0.1 mol dm-3 buffer, unless stated otherwise; [Vitamin B12,] = 6 x niol dm-3.A = Air saturated,f At 35.0 "C. D = degassed.At 45.0 "C.c 0.3 rnol dm-3 buffer.0.075 mol dm-3 buffer.d Calculated from the equilibrium constant and k,.t 0.05 mol dm-3 buffer.0.5 rnol dm-3 buffer1976 1217Kapp.= k,app./k-lspp-l thermodynamically from absorptionspectroscopic data a t different reactant concentrations.Because of the apparently high values of KaPP- and the rapiddecomposition of the vitamin Blz,-~-cysteine complexes a tapproximately equimolecular reactant concentrations, equi-librium constants could not be obtained by the use of theBenesi-Hildebrand or analogous equations.21Values for Kappa were obtained, however, from observingabsorbances due to the vitamin B12,-~-cysteine complex inthe presence of NaCSCN], Na[N,], and imidazole as competi-tors. Typically, 5.0 x mol dm-3 vitamin B12a and3.33 x 10-3 mol dm-3 sodium azide were mixed in thepresence of 0.10 mol dm-3 sodium acetate buffer a t pH 5.5.This solution gave the limiting spectra of the vitamin B12a-sodium azide complex.‘-’ Addition of L-cysteine (1.6 x10-,-5.0 x mol dm-3) caused the absorbance a t 357 nmto decrease.Plots of the left-hand side of equation (6)against [NaN,]/[~-cysteine] allowed the calculation of the[ b zm-Co-N,] - - KaPP.( B12N3) “aN3I (6)[bzni-Co-~-cysteine] KaPP-(B12Cys) [~-cysteine]quotient of the equilibrium constant Knpp.(B12N3)/KBPP*-(B,,Cys). Typical plots of data according to equation (6)are illustrated in Figure 3. Taking a value of 3.2 x lo4dm3 mol-l for Kapp.(B13N3) a t pH 5.5,s a value of 2.4 x lo6dm3 niol-l was obtained for the apparent equilibrium con-stant for the formation of the vitamin B12,~-cysteineadduct.This value was substantiated by carrying outanalogously the competition between L-cysteine and SCN-and imidazole for vitamin BIzs using values of 2 300 and5.5 x lo2 dm3 mol-l for h’aPP.(Bl,SCN) and Kapp-(B12im), andgave the values in 2.10 x lo5 and 2.55 x lo5 dm3 mol-l forKapp-(Bl,Cys), respectively. Agreement among the K*PP.-(B12Cys) values, obtained by the use of three different[Ligandl/[L-Cysteinel800 0 g o 200 LOO 600m I I I I 1 .-/[Ligand I / [ L-CysteinelFIGURE 3 Competition plots for the formation of the vitaminB,,-L-cysteine adduct [equation (6)] a t pH 6.5 using SCN- (O),N,- (A), and imidazole (0) as competitorsligands as competitors, is quite satisfactory and lends cre-dence to this approach, Using values of the kineticallydetermined klaPP* in conjunction with I W P ., values forK-lapp. have been calculated and are given in Table 3.R. Foster, ‘ Organic Charge Transfer Compleses,’ Academic22 G. Jung, E. Breitmaier, and W. Voelter, Ezw. J . Biochem.,Press, New York, 1969.1972, 24, 438.DISCUSSIONAbsorption maxima and absorption coefficients of thethiol complexes of vitamin Biza (Table 1) follow theestablished pattern.lS= As expected for electronegativeligands, the absorption maxima of the y bands for bzm-Co-SHR are at higher wavelengths than that for bzm-Co-OH,. This ‘ atypical ’ spectrum has been taken toindicate the formation of Co-S bonds.15 Lack of reactionof methionine and cystine with vitamin also sub-stantiates this mode of binding.Apparent rate constants for the anation of vitaminBiza by L-cysteine, K1app-, are independent of the hydro-gen-ion concentration.At higher pH values, however,KlaPp. decreases sigmoidally with increasing hydroxide-ionconcentration. The observed pH-rate profile (Figure 4)5rn a”*- 5 tL 5 6 7 8 9 10PHFIGURE 4 pH-rate profile for the interaction of L-cysteine withvitamin B12a in water. Experimental points for k,*PP. are:(O), for air saturated; and (a), for degassed solutions. Thesolid line was calculated from equation (8). Contributionsfrom A,, k,, k,, and k4 are given by lines ( l ) , (2), (3), and (4),respectivelyis explicable in terms of the different reactivities ofbzm-Co-OH, and bzm-Co-OH- and L-cysteine and L-cysteinate ion. Although the sites of protonation ofL-cysteine are not known with ~ e r t a i n t y , ~ ~ .~ ~ the sevenmicroscopic dissociation constants for the pH dependenceof the concentrations of the eight species in solution havebeen ~alculated.,~ This information allows the assess-ment of the concentrations and reactivities of the thioland thiolate anion at different pH values. If L-cystein-ate ions are the only reacting species, values of klaPP.would approach lo9 dm3 mol-l s-l in the pH 3.0-5.0region. In the light of the available data for analogousanations (Klapp. values for a variety of ligands are 10,-lo3 dm3 mol-l s-l) 5-11 such a large rate constant is highlyunlikely. The lack of reactivity of L-methionine, aneutral thiol analogue, can be rationalized in terms ofthe greater steric hindrance encountered in its reactionwith aquocobalamin than that with coba10xime.l~ Athigher pH values, however, bzm-Co-OH, is ionized tobzm-Co-OH-,l-ll and the concentration of L-cysteinateion becomes appreciable.The aquation rates, and hence23 G. Jung, E. Breitmaier, W. A. Gunzler, M. Ottnad, W.Voelter, and L. Flohe, Proc. 16th Conf. German Soc. Biol. Chem.,Tubingen, March 1973, George Thiene Publishers, Stuttgart, 1974.24 R. G. Kallen, J . Amer. Chem. SOC., 1971, 93, 63271218 J.C.S. Daltonthe equilibrium constants for the formation of the L-cysteine complex of vitamin B128, also depend on theprotonation equilibrium bzm-Co-SR- + H+ & bzm-Co-SRH.The scheme describes the complete mechan-ism for the interaction of L-cysteine with vitamin B128.Here k,, k,, k3, and k4 are the pH-independent rateconstants for the formation of the vitamin B,,-~-cysteinecomplex and k,, k-,, k-,, and k-4 are the correspondingaquation rate constants; k5app* is the pHdependent rateKIIimportant path for anation is governed by k,. Titrationof 5.0 x 10-5 mol dm-3 vitamin B,,-L-cysteine complexspectrophotometricaly at 370 nm resulted in a pKIIvalue of 10.9 -+ 0.1.L-Cysteinate ion is 4-5 times more reactive than neutralL-cysteine both with bzm-Co-OH, (K,/k, = 4.3) and withbzm-Co-OH- (k3/k4 = 5.0). Similarly, aquocobalaminis 8-9 times more reactive than hydroxocobalamin bothwith L-cysteine (kl/k4 = 8.8) and with L-cysteinate ionSeveral points emerge from the data in Table 4.ks bzm-Co-OH, + RS- bzm-Co-SR- + H,Obzm-Co-OH, + RSH *, bzm-Co-SHR + H,Ok-1+:! +:\I k, '3bzm-Co-OH- + RS- bzm-Co-SR- + OH-(7)constant for the production of vitamin B1sr.pH-Independent equilibrium constants are defined asKl = k,/k,, K, = k,/k-,, K3 = k,/k,, and K4 = k,/k4.Equation (7) affords the expression of klaPp. a t any pHvalue :(k2/k, = 7.7). Although rate constants for the anationof bzm-Co-OH, are always greater than those for bzm-Co-OH-, binding of L-cysteine to bzm-Co-OH- occursto an appreciable extent. It is recalled that reactions ofimidazole, glycine, azide, and thiocyanate ions occuralmost exclusively with bzm-Co-OH,.l-ll The rateconstant for the aquation of bzm-Co-SR- is similar tothat of bzm-Co-SHR either by water (k-l M k2) or byOH- (k-, M k4).Stability of the vitamin B,,--L-cysteinecomplexes is governed, therefore, by the rate of anationrather than by that of the aquation. The good agree-ment between the K , values determined in the presentwork (1.4 x lo6 dm3 mol-l) and that given in the litera-ture (106 dm3 mol-l) l5 lends credence to our method ofValues of k,, k,, k,, and k4 (Table 4) were calculatedfrom equation (8), by using pK, 7.60 and the appropriateevaluating rate and'equilibrium constants.Activation parameters for the anation were calculatedTABLE 4pH-Independent rate and equilibrium constants for the interaction of L-cysteine with vitamin B,,k, = 11.5 dm3 mol-1 s-1k , = 50 dm3 mol-I s-lk , = 6.5 dm3 mol-l s-lk4 = 1.3 dm3 mo1-l s-lk-, = 5.22 x s - ~k-, = 3.57 x 10-6s-1h-, = 250 dm3 mol-1 s-lk-, = 197 dm3 mol-1 s-lI<, = 2.2 x 106 dm3 mol-lI<, = 1.4 x 106 dm3 niol-lIT, = 2.6 x lo-*K , = 6.6 xthiol concentration.The concentrations of L-cysteineand L-cysteinate were calculated from the microscopicdissociation constants,22-24 assuming that the four formsof the neutral thiols and the four forms of thethiolateions have similar reactivities. Table 4 also containsdata on the pH-independent equilibrium constants, Kl,K,, K,, and K4. Values of K,, K,, K,, and K, wereobtained from the thermodynamically obtained K a p p avalues (Table 3) by means of an equation analogous to(8) which includes [OH-] for K3 and K,.Individualcontributions of k,, k,, k,, and k4 to klaPP- at different pHvalues are given in Figure 4. Below pH 6 the onlyfrom the temperature dependencies of klapp. at pH 5.0and 5.5. At these hydrogen-ion concentrations theoverall reaction is predominantly governed by k,. Theobtained values, AH$ = 14.9 & 0.3 kcal mol-l andA S = -3.6 & 2.0 cal K-l mol-l, resemble closely thosedetermined for the interaction of a variety of ligands withvitamin B12a.10 This is consistent with an S N ~ limitingtype mechanism which involves the dissociation of waterin the rate-limiting step or with outer-sphere complexformation in which a fast exchange occurs.2526 C . H. Langford and H. 13. Gray, ' Ligand SubstitutionProcesses,' W. A. Benjamin, New York, 10651976 1219The prompt formation of vitamin B12r, governed byk,app. in equation (7), only proceeds in the absence of air.This reaction is, therefore, likely to involve free radicalsand it is more complex than is indicated in equation(7) .16 The observed pseudo-first-order rate constantsfor the formation of vitamin B12r from bzm-Co-L-cysteineincrease linearly with increasing L-cysteine concentration.The second-order rate constants for these reactions,k5aPP., were obtained from good straight iines on plottingk, against L-cysteine concentration (Table 3 ) . Valuesof k5aPP- increase exponentially with increasing pH. AtpH < 9, ksaPp. is rate determining. Above pH 9.2,however, kiiaPP* becomes larger than klaPP- and the overallformation of vitamin B12r is increasingly being governedby the rate-limiting formation of bzm-Co-L-cysteine.Lack of data on k p p . for other thiols does not allow acomparison of the relative efficiency of this process.The present work has demonstrated the formation ofvitamin Bl% in the interaction of L-cysteine with vitaminBB~. This finding is significant since it is analogous tothe proposed first step in the enzymatic synthesis ofmethi~nine.~ The spectrophotometric observation ofvitamin Bizr in enzymatic methyl transfer 26 supportsthis postulate. Interactions of other thiols with vitaminBl% and with alkyl cobalamins are being investigatedmechanistically in our laboratories.We thank the National Science Foundation for support.[5/2126 Received, 30th Ocfober, 1976126 H. Riidiger, Eztr. J . Biochem., 1971, 21, 264
ISSN:1477-9226
DOI:10.1039/DT9760001212
出版商:RSC
年代:1976
数据来源: RSC