The nature of anion inhibition of human red cell carbonic anhydrases

ARCHIVES

OF BKMXEMISTRY

AND BIOPHYSICS

Vol. 196, No. 2, September, pp. 501-510, 1979

The Nature

of Anion

Inhibition

THOMAS Department of Pharmacologll

of Human

H. MAREN

Red Cell Carbonic

AND ELSA

Anhydrases

0. COUTO

and Ttirapeutics, Univereitg of Florida Gainesville, Floridu 9.9610

College of Medicine,

Received December 18, 1978; revised February 9, 1979 We studied anionic inhibition of the reaction CO, + OH- e HCO; catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO, as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCOg as the substrate, yielded strict competitive kinetics. Mean inhibition constants, Kr, in the pH range 7.2-7.5 are: iodide, 0.5 rnsf for enzyme B and 16 mru for C; cyanate, 0.8 pM for B and 20 PM for C. When OH- was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (Kt) for cyanate (calculated for infinitely low OH-) is 0.4 pM for enzyme B and 4 pM for C. Apart from the difference in anion affmity and some IO-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site aa Zn-H,O with pK,of about 7,

The mechanism of inorganic anion inhibition of the human red cell carbonic anhydrases has not been analyzed in terms of the several substrates involved and the forward and reverse directions of the reaction. As reviewed by Lindskog (l), there was the suggestion that COZ hydration was noncompetitively inhibited; at that time HCO; dehydration had not been studied. In 1976, we (2) reported that the I,, for anions against human red cell carbonic anhydrase B and C was less (by some 15-fold) for hydration than for dehydration, suggesting different mechanims of inhibition for the two directions. During the ensuing year, our early data led to the statement “that the anion inhibitor mechanism is different between hydration and dehydration when COZ and HCO; are considered as substrates. With I- and CNO- inhibition against both carbonic anhydrase B and C, hydration (COZ) is noncompetitive while that against dehydration (HCO;) is competitive” (3). This led Packer and Tanaka (4) to take up the matter; they reached the same conclusions using Cl- and I- against bovine red cell carbonic anhydrase. In another paper, however, Packer and Bjorkquist reported that azide also gave noncompetitive inhibition 501

of the hydration reaction (bovine enzyme), but mixed inhibition of dehydration (5). We now report the details of our experiments in which I- and CNO- are used against the human red cell carbonic anhydrases B and C. Our earlier conclusion (3) is modified, in that inhibition of hydration appears mixed; dehydration inhibition is classically competitive. The present work makes it possible to assign the true inhibition constants for the anions, from the Iso data of Ref. (2). METHODS CO* hydration was studied by the rate of acidification of barbital buffer. Substrate concentration was varied by the use of tanks of different COz coneentration from 8 to 50%, which furnished substrate in solution at 24°C from 2.8-17.5 mM. These concentrations were determined by direct sampling and manometric analysis and results conform to values demanded by Henry’s Law. The reaction is carried out in a vessel described some years ago (6), which ensures full saturation of the final solution (7 ml) at the admitted gas pressure and easy evacuation and cleaning. Gas flows continuously into the reaction vessel that initially contains inhibitor and enzyme. The usual reaction is started by addition of 2 ml of 25 mM barbital at its pK (7.8) and the time to pH 7.2 is noted. The pH is monitored by a fast reacting electrode GK 0003~9861/79/100501-10$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

MAREN

502

AND COUTO

2322 (The London Company, Cleveland, Ohio) in the solution and Fisher Accumet Model 420 pH/ion meter. Conditions yield initial rates; the uncatalyzed rate is linear with respect to CO, concentration and the pH interval used ensures a sufficiently long reaction time to minimize errors. The rate is calculated from the concentration of buffer acidified (2.1 mM in the pH range cited) per unit time. The pH at which half of the buffer is titrated, 7.53, is used as the pH of the reaction. We found that the hydration rate is not affected by changes in ionic strength, so this was not adjusted. The experimentally determined hydration rate constant is k, in Table I. For study of OH- as substrate, the method was adapted to the pH range 8.0 to 6.7; in these reactions the pH interval was 0.3. HCO; dehydration was measured in a vessel whose bottom is a sintered-glass plate, through which Nz is flushed rapidly and continuously. Phosphate buffer (7.5-S mM final concentration, pH 7.03), enzyme, and inhibitor were added to 5 ml, and the reaction was started by addition of 1 ml NaHCO,, to make a final concentration varying from 15 to 120 mM. The time to reach pH 7.4 was measured and the rate obtained by dividing this into the buffer alkalinized (1.85 mM) in this pH range. Conditions are such that initial rates are measured, product being removed as rapidly as formed. The rate constant is calculated for the mean pH (7.20) of the reaction. We found that variations in ionic strength in the range 0.05 to 0.25 did not affect the dehydration rate constant. It was found that equilibrium was achieved between enzyme and inhibitor without any measurable incubation

time. This is to be expected since both the association and dissociation rates of anions and carbonic anhydrase have half-times well below 1 s (7); our catalytic runs take 4-30 s. Each of the figures shows a representative experiment of three of the same type. The lines were calculated by the method of least squares. The constants obtained for the uncatalyzed reaction and associated values from the literature are given in Table I. Our experiments directly yield k,, km,,, k-,, and Ki. Agreement with the literature is very good including that for K;, obtained from equilibrium measurements. This appears to validate the methods used. K, is taken from the literature using the same correction for ionic strength and temperature that is used for K;. This leads to K, = 10-3.6 M, which is the best conjecture we can make (see Table IV of Ref. (8)). Chemically pure human red cell carbonic anhydrases B and C were obtained commercially (Worthington Chemicals, Freehold, N. J.) or prepared in this department under the supervision of Dr. David Silverman, using affinity gel chromatography as described by Khalifah et al. (9). The molarity of the pure enzyme was determined by the relation (10) that one enzyme unit (the amount that doubles the rate) of C or other high activity enzyme = 1 x 10m9M in the hydration reaction at O”C, using 100% CO*, barbital buffer, and 7-ml reaction volume. The equivalent value for B, obtained by weight or spectrophotometric analysis of the pure enzyme, is 7 x 1O-9 M. All data are for 24°C. The uncatalyzed and catalyzed rate constants are corrected to pH 7.2 (11, 12).

TABLE

H&O,

A

H+ + HCO;

H,O + CO, 9

I

K, =

k, = 0.030

H+ + HCO,

Ia or k-, at given [H+]

k-, km,,

CO, +

K; -

1

10-3.6 M s-’

at pH 7.2 = 0.0033 s-l = &

= 5.2 x

lo4 M-' s-'

H&O, k-,

k

f 10-6.24M: Note [HCO:l rH+] = K; = 10-6.2 M

k-1,

co*

= km, x f$

=

13 s-’

-K = 9.1: Note s = 9.1 H+ 2 k-1 -= k,

K, = 435 K DThe values for kl, k-,,,

k-,

and K’ are from present experiments. Other data from Ref. (8).

ANION

INHIBITION

OF RED

CELL

CARBONIC

RESULTS

Uncatalyxed

Rates (Table I)

In the hydration reaction, uncatalyzed times ranged from 6-34 s as CO, varied from 17.5 to 2.8 mM. The mean k, from several hundred determinations’ was 0.030 2 0.001 s-l. This agrees with earlier data and reviews (1, 8, 11). For dehydration, time ranged from about 5 to 50 s as HCO; varied from 15 to 120 mM. The dehydration rate constant k+, at pH 7.2 was 0.0033 s-l; as shown in Table I this yields the secondorder rate constant kel,, 5.2 x lo4 M-’ s-l. This tallies very closely with earlier data (1, 8, 11). From this we obtain k-, = k-,,.K, = 13 s-l. Data on this constant have been reviewed (l), and our number fits well. Note that the temperature coefficient in this range is 1.1 per degree, so our data corrected to 25°C gives k, = 0.033 s-l and k-, = 14.4 s-l. Thus the k-,lk, ratio, equivalent to CO,/H&O, at equilibrium is 435. Note however that this always depends on knowing K, with more accuracy than is really available (8). These data justify the continuing use of 400 for CO,/H,CO, in physiological work. Catalyzed

Rates

Table II gives the catalytic constants. For hydration we agree closely with Khalifah (12), Wistrand et al. (13), and Steiner et al. (14), and fairly well with Gibbons and Edsall (15). For dehydration there is less relevant literature; our k,,, for C is twothirds that cited (13-15), while K, is close to the mean of other values. Our dehydration values for B are much lower than reported elsewhere for 25°C (13, 15). For keat this can probably be explained by the activating effect of phosphate at 25 mM (16), the concentration previously used (13, 15). At our concentration of 7.5-9.0 mM there is no observed activation (16, 17). Our data fit well with those of Christiansen and Magid 1 This is directly obtained from data at pH 7.5 and below. At pH 8 the rate is approximately 25% greater due to the reaction CO, + OH- ks HCO; for which k,,,, is 8500 M-I s-l (8, 11). Approximate corrections were made for this in the experiments of Fig. ‘7.

503

ANHYDRASES TABLE

II

CATALYTIC CONSTANTSFOR HUMAN REDCELL CARBONICANHYDRASESAT~~~CAND pH 7.2. MEANS 2 SEW = 7) C”

B”

Hydration k,,, s-’ x lO+ K, mM

620 4 62 10 4 1.4

28 k 3 5 k 0.8

Dehydration k&,, s-l x 1O-3 Kh mM

202 2 25 40 -t 3

8 2 0.7 18 k 0.6

n The Haldane relation for C yields 12.3 and for B 12.6.

(17) at 1°C using 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (Hepes) or low PO, buffer. They found k,,, = 3000 s-l and K, = 16 mM. The Haldane relation for ,relating the equilibrium constant to rate constants and K, values in the presence of an enzyme (Table II) yields 12.3 for C and 12.6 for B. Theory for pH 7.2 and K; of 6.2 yields 10. Anion Inhibition Figures l-4 show inhibition by I- and CNO- of human red cell carbonic anhydrases B and C, for both hydration and dehydration. The hydration patterns (left frames) are independent of which anion or isozyme is studied; all yield plots of mixed inhibition. All dehydration patterns (right frames) indicate the same mode of inhibition for both of the anions and both of the isozymes; inhibition is classically competitive. In all of these experiments, each point is the mean of three runs. The lines are drawn by the least squares method and the standard error of K, and V values were 3-20% of their means. For hydration, K1 is calculated for linear mixed-type inhibition from KI =

LA-1

VI

where [A-] is the anion concentration added and V and Vi are the ordinal intercepts of

504

MARENANDCOUTO ENZYME

B Dehydratm

Hydration

FIG. 1. Reciprocal plots of the inhibition by KI of carbonic anhydrase B. E0 = 1.3 x lo-” M for dehydration. In this and Figs. 2-4, mean pH for hydration is 7.53 and for dehydration 7.20.

control and inhibited lines, respectively. cx is the ratio of the dissociation constants of enzyme-substrate-inhibitor complex and enzyme-substrate complex as shown in Fig. 5, following the treatment by Segal(18). Experimentally Q!is the ratio of the abscissa1 intercept of the line for the catalyzed reaction (-l/K,), and the projected abscissal value from the intercept of the lines for catalyzed and inhibited reactions (-l/&J. Thus in Fig. 1, K, = 5 t’tXM and K,i = 10IYkM, whence (I! = 2.

12Ii/

For dehydration, K, was calculated by the usual relations for competitive inhibition. We first get

KfPP =

[A-l --Kini 1 ’ K:,

where [A-] is the anion concentration and KI, and KAi are taken from abscissa1 intercepts of the double-reciprocal plots from control and inhibition data, respectively.

ENZYME

Hydmtmn

Dehydration

II

13rnM I-

10

8

/

02 01

-

mM/sec

6

12 -

4

8

2

4

mlrl/sec L

I /J?Y\

0

01 0.2 03 ht.4

co*

04

[21

0.2 0,

0

01 .02 'ht.4

04

06 07

tico;

FIG. 2. As Fig. 1, but for carbonic anhydrase C. E, = 1.1 x 10m9M for hydration and 2 x 10m9M for dehydration.

ANION

INHIBITION

OF RED CELL CARBONIC ENZYME

ANHYDRASES

505

B

Hydratm

Dehydmtm

IO

0

0.5 0.4 0.3 0.2 0.1

I4rut.d CNO-

0 OJ 0.2 0.3 0.4

04

02

0

01

02

‘/mm co,

.04

06

‘/mM HCO;

FIG. 3. Reciprocal plots of the inhibition by KCNO of carbonic anhydrase B. E, = 1.5 x 1O-8 M for hydration and 5.3 -X lo-* M for dehydration.

Primes are used to denote dehydration constants. Equation [2] is used for the system involving phosphate buffer, and its affinity for these enzymes, particularly B, must be taken into account.2 We determined the K, of phosphate independently in the hydration reaction using barbital buffer; Eq. [l] yielded 2.3 mM for B and 28 mM for C. This agrees with Ref. (2) and data cited there. The true K,‘s of the anions are now obtained by the relation

usually 7.5 mM, and the K,‘s for phosphate as given just above. Table III shows the values for K,. For iodide there is remarkably good agreement between hydration and dehydration numbers: for CNO- there is nearly a 3-fold random range. To test further the postulated mechanisms and gain additional data for CNO-, the experiment of Fig. 6, generating a Dixon plot, was done. Conditions were as described above for the dehydration reaction, except that it was done at 2°C to lower the uncatalyzed rate, and mean pH KfPP was 7.0. k,,, is 2540 s-l, and K, about K, = [31 40 mM, fair agreement with data of Refs. 1 + F’hosl ’ (16) and (17) for similar conditions. Figure 6 K I phos. shows simple linear competitive inhibition. where KfpP is from Eq. [2], [Phos] buffer

2 The choice of buffers in this work is worth comment. Barbital in the concentrations used is ideal, as it is does not inhibit the enzyme, does not stimulate the enzyme, does not affect the uncatalyzed reaction. Unfortunately its physical surface activity leads to foaming in the dehydration reaction and it could not be used. We turned to low concentrations of phosphate, which do not activate enzyme (16, 17) or affect the uncatalyzed rate. There is however, considerable inhibition of carbonic anhydrase B and this is taken into account (Eq. [3]) in the calculation. Note however that our conclusions regarding inhibition mechanism do not seem to be endangered by this, since the mechanism for carbonic anhydrase C, which is but slightly affected by phosphate, is identical to that of B.

TABLE

III

CALCULATEDK~OFI-ANDCNO-AGAINST CARBONICANHYDRASES

Dehydratior?

Hydration” B ICNOCNO-, KP

0.5mM

B ANDC

C

14 mhi 1.2 /.bM 10 w 0.4 /.&Mb 4 /.LMb

B

0.5mM 0.5 PM

C 18mM 29/.&M

a Calculated for linear mixed-type inhibition, Eq. 1. b Calculated for competitive inhibition against HCO; (dehydration) or OH- (hydration), Eqs. [2] or [3].

MAREN

506

AND COUTO

ENZYME

C Dehydration

Hydrotlon

f/mM CO2

VmM HCO;

FIG. 4. As Fig. 3 but for carbonic anhydrase C. E, = 6 x lo-lo M for dehydration and 2 for dehydration.

At 3’7 PM cyanate the catalytic reaction has been reduced to 5% of normal; this is the methodological limit since at this point the uncatalyzed reaction dominates the total rate (by 5- to lo-fold) and the residual inhibited catalytic rate is virtually undetectable. The plot yields a KaPP of 1 PM for CNO vs enzyme B. Correction for phosphate according to Eq. [3] yields K, = 0.3 /.LM. ,

KS CO2

+ EOH- e H’jfOH-

co2

KS

EOH-CO2

K.

+

JI

E e

EC02

+

+

A11% EA-

+

F==+

EHCO;

F===

E + HCO; +

IKI EA-

~

CO2 7

L EC02A-

FIG. 5. A model for catalysis and anion inhibition in the CO,-carbonic anhydrase system. K, and K: are shown as dissociation constants; it is not certain whether the measured Michaelis constants are equivalent to dissociation constants. We assume that (Y = K,,IK, = KEC,,zA-IKE,,HC02, where the dissociation is to substrate. As shown, K, applies to a given pH. The relationships also hold for K’j, the dissociation constant of EA- at zero OH-; (Y= KECOIA--IKEA= K&2A-IK’&-.

Effect of pH upon Anion Hydration Reaction

x

Inhibition

1OV M

in the

Since (OH-) is a ligand of the enzyme(s) (l), it is important to consider the true affinity of the anions vanishingly low (OH-). Figure ‘7 shows reciprocal plots of rate versus (OH-) for the catalyzed hydration reactions, normal and inhibited by CNO-. The control data show the expected relation that increasing (OH-) increases rate, to the degree expected if the active enzymes for this reaction are basic with pK, of about 7.0 (1, 19). Cyanate yields competitive plots, from which the true dissociation constant, designated KY, may be calculated from the type Eq. [2]. (KP is now given directly, not Kg”“, since there is no correction for buffer binding in the hydration reaction.) This yields 0.4 j.kM for C. On this basis the higher KI values obtained above from Figs. 3 and 4 are subject to the important modification that they were determined at a fixed OH- concentration, and competition with OH- was not calculated. Values are compared in Table III. Chloride was studied against enzyme B and yielded similar plots to Fig. 7. The KP calculated using OH- as substrate was 7 ITIM.

ANION INHIBITION

OF RED CELL CARBONIC

ANHYDRASES

507

1500 I

pt.4

CNO-

FIG. 6. Dixon plot of cyanate inhibition of human red cell carbonic anhydrase B catalysis of HCO, dehydration. T = 2”. [E,] = 1.3 X lo-* M. Mean pH 7.0.

Note that the observed K, for OH- is anion binding is restricted to the acidic related to the K, of the enzymes by the species of the enzyme, and of enzyme-CO, expression K, = K, /K, , whence p K, for C complex (Fig. 5). is 6.8 and for B 7.0. Complete pH versus rate profiles for DISCUSSION these enzymes suggested a pK, close to 7 for C, and that for B, 0.6 units higher (12). The data are clear in showing that anions Figure 7 suggests competition between compete with OH- (in the hydration reaction) anions and OH- at the active site, or the and with HCO; (in dehydration) for the more formal general interpretation that active site of carbonic anhydrase. This

ENZYME

B

yf.

CNO-

2.qLM

40.

&&

m‘PM zo-

FIG. ‘7. The figure shows the competition between CNO- and OH- for carbonic anhydrases B (E, = 7 x 10-S M) and C (E, = 4 x 10-l“ M). CO* = 5.6 mM. Barbital buffer mixtures used; mean OHcalculated from midpoint of titration in the 0.3 unit reaction interval.

508

MAREN

AND GOUT0

latter agrees with Packer and Tanaka for Cl- and I- inhibition (4), but not with Packer and Bjorkquist for azide (5). Earlier work by Magid is difficult to interpret, since there were different patterns for the several anions and isozymes (16). When COZ is the substrate, inhibition by the anions appears mixed. This differs from our preliminary comment (2) and from Packer and Tanaka (4) in which hydration was reported as noncompetitive. It is emphasized that the present experiments give the same result when V is plotted against V/S, and also that the lines of Figs. l-4 are substantially unchanged when replotted minimizing the weighted sums of the square on the assumption that each velocity measurement has the same standard error (20). Our result is perhaps not surprising. Although it has not been considered in carbonic anhydrase kinetics, careful critics of enzymology doubt if there is in reality a pure noncompetitive case (20). It is interesting that Davis (21) said that SHwas noncompetitive in the hydration reaction against the human enzyme preparation (before there were known to be isozymes) but recalculation and plotting of his data minimizing the weighted sum of squares (20) does not yield a clear pattern. Lindskog’s data on Cl-, I-, and NO; against the bovine cobalt enzyme agree with ours, in showing that anions displace both K, and k,,, at pH 7.0 (22). Our data are not complicated by slow dissociation of the EI complex; it may be surmised from Taylor and Burgen (7) that this occurs in
Our finding that anions appear more active (i.e., lower I,,,) when CO, is substrate than HCOB-, under conditions of near substrate saturation (2), is readily explained by the different modes of inhibition in the two sets of reactions. We may now calculate from these data (2) the KI in the dehydration and hydration reactions. For dehydration we first calculate an apparent KI from the relation

Ky

=

Kh K:, + S

.I 50*

[41

(S) in those experiments was 30 mM HCO;; Kh is given for each enzyme in Table II. Since 9 mM phosphate3 was used in these experiments, KrPP must be modified by Eq. [3] above to yield the K, for the various anions. The K, for phosphate is as given above, 2.3 mM for B and 28 mM for C. We now have a general relation between the observed Iso and the K, for all the anions, such that for enzyme B K, = (0.07) Iso, and for C K, = (0.40) 150. For hydration, the results for S = 5 mM and 50% inhibition (2) can be introduced into Eq. [l]. When this is done, for (Y = 2, V/Vi = 1.5, and K, = Iso. As shown above (I! is approximately 2, but it is evident that only small errors are introduced if the range is 1.5-2.5. We therefore take the observed I5o for the hydration case of Ref. (2) as the K,. These calculations yield a series of K, values for Cl-, Br-, acetate, NO;, C104, and CNS for the hydration and dehydration runs, for both enzymes, based on the I,, values of Ref. (2). These are given in Table IV, including the earlier data on I- and CNO-. There is remarkably close agreement between the hydration and dehydration K,. This agrees with present direct findings with I- and CNO- (Table III). We confirm earlier findings (2) that anion affinity for enzyme B is generally much greater than for C; this difference remains unexplained. The only exception is ClO; which does not discriminate between B and 3 The inhibitory effect of phosphate in the dehydration reaction was not observed experimentally (2); this must be related to the curious activating effect it has in the catalytic dehydration of B (1’7):See Footnote 2.

ANION INHIBITION

OF RED CELL CARBONIC TABLE

K,

(mM)

OF

IV

ANIONS FOR HUMAN RED CELL CARBONIC ANHYDRASES B AND Ca B

ClBrICH,COONO; ClO, CNS CNO

509

ANHYDRASES

C

Hydration

Dehydration

6 4 0.3 7 7 3.6 0.2 0.0007

4 0.4 11 6 1 0.1 0.0005

7

Hydration

Dehydration

200 200 26 70 35 1.3 0.6 0.02

240 240 46 53 36 2 1.8 0.05

(1From data of Ref. (2). Hydration numbers are equivalent to Is0. Dehydration calculated as in present text.

C (Table IV). Of interest is the finding that within the halions discrimination rises with molecular size, reaching 100 for the ratio of activity (B/C) of iodide. Roughly, however, the order of inhibition of anions against the two isozymes is similar, with Cl- being the least, and CNO- the most active. This order of activity of anions (2) corresponds to the so-called lyotropic or Hofmeister series, which is related to the size and hydration of the anions, and their effects upon the structure of water. The order of decreasing negative hydration energies, F- > Cl- > Br- > I- > ClO;, corresponds fairly well to increasing activity against the carbonic anhydrases, as well as certain other biological systems, notably thyroid (23). It is likely that these various facts are related, since the active site of this enzyme is Zn-bound water. A clarification of the problem in quantitative terms should reveal much about the enzymic mechanism. In this connection further pharmacological work on CNO-, the most active anion, appears worthwhile; its KP (0.4 pM) against B approaches that of the K, for OH- at the active site (0.1 PM). 4 Fluoride does not inhibit either isozyme below 366 mM (2), and to find if there is any activity at all would require further studies with careful attention to ionic strength. From what has been done, there seems no discrimination between B and C.

ACKNOWLEDGMENTS We are particularly grateful to our colleague Dr. David N. Silverman for his close study of these data and discussions which have led to the ideas presented here. We thank Dr. Gautam Sanyai for help with calculations and for carrying out the experiment of Fig. 6. Work supported in part by NIH Grant EY-02227. REFERENCES 1.

2. 3. 4. 5. 6.

LINDSKOG, S., HENDERSON, L. E., KANNAN, K. K., LILJAS, A., NYMAN, P. O., AND STRANDBERG, B. (1971) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 5, pp. 537-665, Academic Press, New York. MAREN, T. H., RAYBURN, C. S., AND LIDDELL, N. E. (1976) Science 191, 469-472. MAREN, T. H. (1976) Science 194, 745-747. POCKER, Y., AND TANAKA, N. (1978) Science 199, 907-969. POCKER, Y., AND BJORKQUIST, D. W. (1977) Biochemistry 16, 5693-5767. MAREN, T. H., ASH, V. I., AND BAILEY, E. M., Jr. (1954) Bull. Johns Hopkins Hosp. 95, 244-255. TAYLOR, P. W.., AND BURGEN, A. S. V. (1971) Biochemistry

10, 38594866.

EDSALL, J. T. (1969) in COz: Chemical, Bio chemical, and Physiological Aspects (Forster, R. E., Edsall, J. T., Otis, A. B., and Roughton, F. J. W., eds.), pp. 15-27, National Aeronautics and Space Administration, U. S. Government Printing Office, Washington, D. C. KHALIFAH, R. G., STRADER, D. J., BRYANT, D. H., and GIBSON, S. (1977) Biochemistry 16. 2241-2247.

510

MARENANDCOUTO

10. MAREN, M. N.

T. H., (1960)

PARCELL, J. Pharmucol.

A. L., Exp.

AND MALIK, Ther. 130,

389-400. 11. KERN, D. M. (196O)J. Chem. Educ. 37, 14-23. 12. KHALIFAH, R. G. (1971) J. Biol. Chem. 246, 2561-2573. 13. WISTRAND, P. J., LINDAHL, S., AND WAH~RAND, T. (1975) Eur. J. Biochem. 57, 189-195. 14. STEINER, H., JOHNSON, B. H., AND LINDSKOG, S. (1975) Eur. J. Biochem. 59, 253-259. 15. GIBBONS, B. H., AND EDSALL, J. T. (1964) J. Biol. Chem. 239, 2539-2544. 16. MAGID, E. (1968) Biochim. Biophys. Acta 151, 236-244.

17. CHRISTIANSEN, E., AND MAGID, E. Biochim. Biophys. Actu 220, 630432.

(1970)

18. SEGEL, New

Wiley,

I. H. York.

(1977)

Enzyme

19. KERNOHAN, J. C. (1965) Biochim. 96, 304-317.

Kinetics, Biophys.

20. CORNISH-BOWDEN, A. (1976) Principles Kinetics, Butterworths, London. 21. DAVIS, R. P. (1959) 5674-5678. 22. LINDSKOG, 23. WRIGHT, Physiol.

S. (1966)

J. Amer. Biochemistry

E. M., AND DIAMOND, Rev. 57, 109-156.

Acta

of Enzyme

Chem.

Sot.

81,

5, 2641-2645. J.

M.

(1977)