Robert S. Mc(1uate1 a n d J o h n E. R e a r d o n Willamette University Salem. Oregon 97301
Kinetics of Formation of Cobalt(l1)- and Nickel(ll)Carbonic Anhydrase
T h e essential role that metal ions play in certain biological systems was established previously h y considering t h e hydrolvtic zinciI1)-containinr metalloenzvme. carhonic anhvdrase ( 1 ). T h e metal-free &enzyme can be prepared with little difficulty, and several other metal ions have been found t o coordinate a t t h e site vacated by t h e zinc ion. When examining t h e interactions of several different metal ions with apocarbonic anhydrase, t h e significance of kinetic regulation of hoth formation a n d dissociation of metallucarbonic anhydrase species surfaced ( I ) , and it. is t h e kinetic hehaviorassociated with the formation of two metallocarbonic anhydrase species, t h e biochemically active Co(I1)- and t h e inactive Ni(ll)- derivatives, t h a t serves a s t h e focus of this article. Because carbonic anhydrase is a well-behaved enzyme, it has been subjected to several kinetic investigations during the past decade with t h e expectation of providing a moredetailed understanding of t h e metal ion-protein interactions t h a t are vital t o t h e proper functioning of t h e native zinc(I1)carbonic anhydrase (2-6). T a h l e 1 contains a summary of pertinent kinetic results. Based on these kinetic studies, several interesting features t h a t are unique t o metalloprotein chemistry have been uncovered. a n d these asnects are germane t o our general understanding of bioinorganic chemistry. T h e experimentation involved can he readily adapted t o allow upper level undergraduate students t o gain experience not only in handling and studying enzymes, h u t also in acquiring kinetic data, analyzing these data for rate constants, and determining rate laws from these d a t a for seemingly complex biochemical systems. Such laboratory activities can be performed a s p a r t of a n Inorganic Chemistry course o r in conjunction with a n integrated-laboratory Experimental Section In order to examine the kinetics of M(II)CA furmation (where M = Co or Ni), apocarhonic anhydrase (apoCA) must he available. Ro. vine apoCA can he prepared asdescrihed previously ( I ) .'The residual phen and acetate in the apaCA solution must he removed hy dialysis againstseveralchangesof cold ( 0 4 W pH 5.5 MES huffer (1.0 X 10-' M ) containing 0.10 M KCI. The huffer solution was freed of extraneous transition metal ions hy employing the dithizane-CC14 extraction (1I. Aqueous stock solutions of Cu(NOplz,Ni(NO:h, and Zn(NOJ2were prepared from reagent grade chemicals and deionized water, and they were standardized according to conventional methods (81. The solutions were found to have the following concentrations: Caz+ (0.413 M), Ni2+ (0.0992 M). and Zn2+ (0.161 M). Formation Kinetics for Co(1IjCA A compilation of appropriate experimental conditions employed in studying the formation of Co(ll)CA appears in Tahle 2. A representative experiment conforming to eqn. (1) consists of the following.
300 pl of 3.3 X 1 0 P M apoCA at pH 5.5 were delivered into ahnut 4.5 ml of extracted pH 5.5 huffer (10VM MES with 0.10M KCI) in a 5.00 ml volumetric flask. The flask had heen presoaked in a saturated Na2ED1'A solution, rinsed thoroughly with deionized water, and air dried. Thecontents were thermostatted at 25.0% in a water bath. The volume was then adjusted to 5.00 ml with additional huffer. When ready to initiate the reaction, a known volume of standard Co" so-
Table 1. Kinetic Parameters tor Metallocarbonlc Anhydrase Species at 25"Ca
J@ (kcal
kf (M-' MZ+ "02+
Gu2+ Zn2+
Cd2+ CO~+
Mn2+ NiZt
%-') --pH
-108 a 5.6 X lo3 2.1 X lo4 4.0 X lo3 530 81 7.4 2.9 56 8.5 22
0.61
--
7.9 5.30 7.56 5.5 5.0 7.5 6.10 5.5 7.5 6.2 7.00 5.50
AS* (cal
mole-' --
mole-')
...
23
OK-')
Refer-
ence -. .
...
(4)
38 30
(3) (8
21 ... 24
.. .
.. .
...
39
( 3)
26
38
(3)
.. .
... ... .. . . .. ~
-
.. . ... ... ... ...
cn
(61 (7) .--
Rater vary with ionic strength: values not neceswily at ram ionic strengths. a Calculated from h, = k,K,.
lution, for example 15 PI, is quickly delivered from a micmpipet.gising a total, initial vdume of 5.015 ml. The quantity of Co2+added should he such that there is sufficient excess Co'+ over apoCA to conform to pseudo first-order conditions. Once thr Co2+is added, the contents are immediately hut gently agitated prior to thermostatting at 25.0% 500 p1 aliquots of the reaction mixture were removedperiodically and assayed for enzymatic activity by using the synthetic substrate, pnitrophenylacetate.%ince apoCA is biochemically inactive and the product Co(1I)CA is active, the rate of reaction can he monitored by ohs~rvingthe increase in the p-NPA activitv with time. As thisreaetion proceeds, more CollllCA forms, and the assay solutions show increased activity. Figure l a illustrates the type of kinetic data gathered. Additional runs, where the initial Co'+ concentration is varied, are recommended. Since experiments are performed under pseudo first-order conditions, the pseudo first-order rate constants, k,,m, can he obtained from the negative slope of the straight line which results from the plot of ln(a., - a ) versus time (where o = relative activity or relative slope). Note that the k,,h,valuexare independent of the apoCA concentration. Formation Kinetics for NfllIjCA
Experimental details used in studying the formation kinetics of Ni(ll1CA also appear in Tahle 2. The experimental approach parallels the procedure described for Co(l1)CA with two important differences. One, different concentrations of Nizt are used because the Ni2+ reactions are inherently slower than those withCo2+.Serond. Ni(1l)CA is an inactive enzyme, and since hoth reactants and products are hiochemically inactive, an indirect method of monitoring the kinelics
'
Author to whom corresnondence should he addresed at Fmd and Drug Administration, Rur& of Foods (HFF-335). ZOO.'(>" St. S.W.. Washington, D.C. 20204. The 5 0 0 ~ aliquot 1 of the reaction mixture was added to a 1.00 em spectrophotometric cell containing 1.70 ml deionized H20, 0.30 m1 0.10 M Hepes at pH 7.1, and 1.00 ml of p-NPA suhstrate (prepared as described in reference ( I )). Once the aliquot. is added Lo the assay solution, 45 s or less is sufficient for a satisfactory reading. The re& tion between Co" and apoCA does not., in an ahsolute sense, stop during the time needed for the assay. Hecause of the dilution of reactants by a factor hetween 6 and 8'12 upon addition of the aliquot to the assay solution, however, the rate of continued formation of Co(ll1CA during theactual assay is reduced sufficiently tc allow the system to he treated as if actual quenching had imorred. The times noted for each assay are determined when t h aliquot ~ is introduced into the assav solution. Volume 55. Number 9. September 1978 1 607
. .
must he emoloved. An aliauut of Zn2+ must he added t o each spectrophotometric cell eontaming the assay solution. A 5 pl aliquot of n lR1 M Zn2+ npr 2 00 ml of assav solution has been found to be suf-
effect of rapidly quenching the reaction by hinding to any unreaded apoCA, thereby generating the active ZnII1)CA.
-
Nix* + apoCA (inactive)
ht
Ni(1I)CA (inactive)
(2)
(fZn" Zn(I1)CA fast (active)
This Zn(1I)CA subsequently catalyzes the hydrolysis of p-NPA. At early times during a reaction, relatively little apoCA has been consumed by reaction with Ni2+, so a large activity (steep slope) will be observed. At longer times, moreof the spoCA has reacted with NiPt, so the observed p-NPA activity will be smaller. Figure l b shows the typical response for such a reaction. Again pseudo first-order conditions prevailed where NiZ+ was present in excess, and standard first-order plots using relative activity measurements (slopes) as a function of time wereused to obtain the hob, values. The pH of each solution was measured a t the conclusion of each experiment by using a Cornine semimicroelectrode model 416050 with a Beckman Zero-
Results and Discussion For a constant pH, the simplest rate law that one could propose to describe the formation of M(1I)CA from aqueous M2+ and apoCA appears below.
rate = d["(ll)CA1 = ki[M2+l[apOCA~ 13) dt Since the reverse reaction, the uncatalyzed dissociation of M(1I)CA into M2+ and apoCA, is generally very slow (2,6), including a term for the reverse process was found to he unnecessary. With asufficient excess of MZ+over apoCA (pseudo first order conditions), the rate law simplifies to rate = k,b[apoCA] where (4) hobs = ki[MZCI A plot of k , ~ v e r s u s[MZf] should giveastraight line with the origin as the intercept and k, from the slope if the proposed rate law is valid.
Figure 1.p-NPA ASSaySas a Function of Time for M(I1)CA Formation (a) Co(1l)CA formation: [CoZ+Ii = 1.24X M [apoCA],= 1.95X 10-5M: pH= 5.49. Aaivity develops as a function of time (increasing slopes) asthe aniva W1l)CA forms. (b) Ni(1I)CA fwmation: [Nizt]i = 1.94 X lo-' M, [apaCAIi = 1.91 X 10P M, pH = 5.46. Activity drops as a function of time (decreasing slopes) as the inactive Ni(1I)CA forms (see text for explanation).
Care must be exercised in preparing the spedrophotometric cells with the desired solutions. Sufficient ZnZ+must be added to the cell toconsume all apoCA that has not coordinated to Ni2+.The addition of too much Zn2+may result in the displacement of Ni2+ from Ni(11)CA by Zn2+. Zn2+ Levels close to those specified are recommended. Also see reference (6).
Table 2. S u m m a r y of Experimental Data tor Kinetic Trialsa Formation of Co(ll)CAb "01 VOI apoCAb(lrl) Co2+ *(&I)
300 300 300 300
[~POCA], ( M X lo5)
5.002 5.005 5.010 5.015
1.95 1.95 1.95 1.95
total volume of system (ml)
[awCAl, ( M X lo6)
5.050 5.100 5.200 5.250 5.300 5.400
1.29 1.91 1.25 2.48 1.23 1.61
2 5 10 15
Formation of Ni(1i)CA' YOI "01 ,42+ I a w C A b (ul)
200 300 200 400 200 300
total volume of system (ml)
50 100 200 250 300 400
[Co2+1,
"01 aliquot per assay (&I)
pH
(sec-' X lo3)
1.65 4.13 6.24 12.4
500 400 600 500
5.50 5.50 5.49 5.49
0.696 1.86 3.44 5.16
[Ni2+], (MX lo3)
"01 aliquot per assay (@I)
pH
500 400 300 300 300 500
5.45 5.46 5.55 5.45 5.42 5.44
( M X lo4)
0.980 1.94 3.61 4.72 5.60 7.35
16br
k*, (sec-' X 107
All runs performedat 25.0-t 01°C in 0.10MKCi.
.me assay solution. pisced in a loo cm spectrophotomelricsell, conrirtr of 1.70 ml of deionized n20.0.30ml0.10M H W ~ aSt pn 7.1,and 1.00ml pNPA solution
'Concenvationof stack apoCA = 3.25X lo-'
M.
CConcentration at stock Co2+ = 0.413 M. -meassay solution is the same as noted in (b) with me amtion of 5 p1 of fCancentrstionof stock Ni2+= 0.0992 M.
608 / Journal of Chemical Education
0.161 MZ++
per speckophotdric cell.
0.479 1.04 2.03 2.34 3.39 4.64
nitude) in rates of formation of M(1I)CA. While the reason(s) for such a wide variation are unknown a t the present time, it is known that the rates of formation between aquated bivalent metal ions and apocarbonie anhydrase are significantly slower by a factor of 100 or more than the corresponding rates of formation of bivalent metal ion complexes of smaller polydentate ligands such as 1,lO-phenanthroline and 2.2'2''-terovridine (2. 9. 10). The reduced formation rates for . rncr.4lucarlwn~~ anhydraics arc cms~stcntw t h the imnrually large activatiun enthalpiti 181ahour 2 1 kr;d mule, n i i~pptwdto \,aIues of 5 IS kral mole for the more rlars~calpulydrntm~hrantls. I'an~cularly large and pusltwe n~.twiatitmentropi~s01.11 PO tcomp~rrdwit h m d l . necatwe valws tor thes~mplepulylcntate Ilynnd.) rend toportially ~,flsctthe unfavmshle AH:.The Inrw AS' value* in the ~ n j t e i n systems are ascribed to the physically t i w e complex coordination site for the metal ions. The bindine site is buried nearlv 15 A within the
.
Figure 2. k*
versus [M2+] for Co(l1)CAand
NI(I1)CA at pH 5.5.
Figure 2 contains these plots, providing experimental support for the above interoretation. The kr values of 4.1 f 0.1 M-' see-' and 0.6 i 0.3 A { - I qrc'-l for rl~crohalrillland nickel(l1r systems, reipectivrly. are linear lrasr squares rewlls. The largrr scatter in theexperimental poinrs for the NI~IIK'Asysrrm result. from the indirect methud of monitoring the kinetics. Rudolf examined the kinetics of formation of Co(1I)CA as a funetion of pH using slightly different experimental conditions than were used in this study. The pure B isozyme and an ionic strength of 0.035 was used hv Rudolf (3). while the A and B isozvme mixture and an ionic strength of 0.10 were employed in these ixperiments. The kf value for Co(II)CA of 4.1 i 0.1 M-'sec-' is in relatively good agreement with the value of 2.9 M-' s ~ c - ' extrapdated t o pH 5.5 from Rudolf's work. No literature value is available for a comparison of the experimental kf of 0.6 i 0.3 M-' sec-I for Ni(1I)CA a t pH 5.5. This value is consistent with the observation that Ni(1I)CA forms more slowly than Co(I1)CAunder identical conditions of temperature, pH, and reactant concentrations. Examination of the k, values for several metallocarbonic anhydrases in Table 1 reveals a hroad range (covering four orders of mag-
those from within the apoenzyme cavity in forming the activated complex.
Acknowledgment Acknowltdcment is made to the D m m uf The Pernkum Revarch Fund t h n u ~ hgrant *YOII-R.{. ndmmtstpred 11). the American ('hsmical Soclet), fur thp part~nlsuppalrt uf t h ~ study. s
Literature Cited !I1
MeQuatr R.S.. J.CHEM. EDUC..54,645
(19771.
1%) Henken8.R. W.and Sturteuant.d. M.. J. Am. ?hem Sor.. NI.2669W681.
(31 Rudo1f.S. A..Ph.D. T h a ~ i r Yale . University. 1971. (41 Fitzgarald.J.J. and Chasteen. N. D., Riochmnirlr.~. 13, G I 8 119741. 151 Gwber.K., Ng, F.T.T..and Wilkins, R.G.,Rioinnrn. Cham., 4,158(19751. 161 Wilkins,R.G.snd Wi1liama.K. R.. J. Am. C h e m So?. 96.Z241119741. (71 McQuatP, R. S. and Reardon, J. E., to he puhliahed. 181 Sehwartlenbaeh, G . and Flmhka,H.. "CumplexometrieTiUatinns,"2nd ed.. Methewn and Co.. Ltd., London. 1969, pp. 242-9.260-5. (91 Wilkins,R.G.,PumAppl.Ch~m..31,58:4!197:41. (101 Coleman, .I. E., Inorganic Riarhemisln. 1.506 119731.
Volume 55. Number 9. September 1978 / 609
