The Nature of Binding of Competitive Inhibitors

to Alcohol Dehydrogenases”

(Received for publication, December 28, 1970)

J. MAITLAND YOVSG$ AND JUI H. WAKG

From Kline Chemistry Laboratory, Yale Univemity, Nex Haven, Connecticut 06530

SUMMARY

Two classes of metal ion binding sites in horse liver alcohol dehydrogenase are distinguished by the observed rates of replacement of zinc by cobalt in acetate and in Z-(N-morpho- line)-ethane sulfonic acid buffers. Hybrid enzymes contain- ing both zinc and cobalt metal ions have been prepared and exhibit visible absorption maxima at 655 nm and 740 nm.

Binding of azide, a substrate-competitive inhibitor, to yeast alcohol dehydrogenase and to native and metallo hybrid horse liver alcohol dehydrogenases has been investigated. The infrared absorption maximum for azide bound to yeast alcohol dehydrogenase in the presence of NAD+ is 2070 cm-l, and that for azide bound to native horse liver alcohol dehydro- genase is 2065 cm-l. The frequency of azide in a solution of a hybrid liver enzyme is within experimental error equal to that observed upon binding to the zinc enzyme. Kinetic analysis also indicates that the binding of azide to zinc and hybrid liver enzymes is similar. No change is observed in the visible spectrum of the hybrid enzyme upon binding of azide.

No change in the visible spectrum is observed upon binding of pyrazole, a neutral substrate-competitive inhibitor, to hy- brid horse liver alcohol dehydrogenase. Stopped flow measurements show that the rate of binding of pyrazole to the hybrid enzyme is at least as great as, and perhaps greater by a factor of two than, binding to the native en- zyme. Similarly, 1, IO-phenanthroline binds twice as fast to the hybrid enzyme as to the zinc enzyme, and no change in the visible spectrum is observed upon binding of this co- enzyme-competitive inhibitor.

These results may be interpreted to indicate that none of these inhibitors binds to the “easily exchangeable” metal ions in horse liver alcohol dehydrogenase.

The elucidation of the roles of metal ions in alcohol dehydro- genases has been greatly advanced by the preparation of cobalt-

* This investigation was supported in part by Research Grant GM-04483 from the United States Public Health Service.

1 National Science Foundation Predoctoral Fellow, 19G6 through 1970. This work is taken from the dissertation to be submitted to Yale Universitv in martial fulfillment for the Ph.D degree. Present address, &par&ent of Chemistry, Bryn Maw-r College, Bryn Mawr, Pennsylvania 19010.

substituted and cadmium-substituted horse liver alcohol dehy- drogenase (1, 2), and of yeast alcohol dehydrogenase which contains cobalt (3). Zinc is essential for the catalytic activity of the native forms of both alcohol dehydrogenases (4-7). The ex- tensive studies of Drum, Li, and Vallee (8) and Drum et al. (9) have shown that enzymatic activity correlates closely with the zinc metal ion content of two “functional” metal ion binding sites per mole of horse liver alcohol dehydrogenase, and the remaining zinc ions serve a structural role. Recent spectroscopic studies by Drum and Vallee (10) have indicated that only two of the zinc ions in the liver enzyme interact with 1 ,lO-phenanthroline and 2,2’-bipyridine, thus demonstrating the differential chemical reactivities of the zinc ions.

1 , 10.Phenanthroline is kinetically competitive with the co- enzyme, NADH (11, la), and the observed spectroscopic changes (13-15) indicated that 2 molecules of 1, IO-phenanthroline inter- act with zinc atoms of horse liver alcohol dehydrogenase. Re- cent stopped flow studies by Gilleland and Shore (16) of 2,2’- bipyridine binding have been interpreted as indicating the involvement of zinc in the liver alcohol dehydrogenase. Pyraz- ole, another ligand for zinc, is an inhibitor of the liver enzyme (17) and the yeast enzyme (18) which is kinetically competitive with substrate. The rate of binding of pyrazole to horse liver a,lcohol dehydrogenase in the presence of NAD+ has also been interpreted as indicating the involvement of zinc (16).

In order to gain further insight into the roles of the metal ions, we have examined the effects of neutral and anionic inhibitors on the enzymatic activity and the spectral properties of various alcohol dehydrogenases and have determined the rates of bind- ing of inhibitors to these enzymes. Studies of the rates of re- placement of zinc ions of horse liver alcohol dehydrogenase by cobalt have enabled us to prepare hybrid enzymes containing both zinc and cobalt. Characterization of these hybrid enzymes has provided additional information on the differences in chemi- cal reactivities of the metal ions in horse liver alcohol dehydro- genase.

EXPERIMEh-TAL PROCEDURES

Horse liver alcohol dehydrogenase, lots 9GA and 8JA, was purchased from Worthington Biochemicals. Crystalline sus- pensions in 0.02 M phosphate buffer, pH 7.0, containing 10% ethanol, were dialyzed against large quantities of buffer before use in kinetic inhibitor binding studies. Enzyme concentrations were routinely determined by absorbance at 280 nm, using the

2815

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2816 Binding of Competitive Inhibitors to Alcohol Dehydrogenases Vol. 246, No. 9

^. \ u.l ’

10 30 HSOOIJRS

70 90

FIG. 1. Cobalt replacement of zinc in horse liver alcohol dehy- drogenase. The reaction is followed in terms of the fraction of 3.7 g atoms of zinc per mole of enzyme not exchanged, as discussed previously (8). Experimental conditions were presented in Table I. 0, Experiment 1; A, Experiment 2; 0, Experiment 3; 0, Experiment 4.

specific absorptivity 0.45 mg-* cm2 (19). Concentrations of yeast alcohol dehydrogenase (lots 7DB and 9 GE, Worthington) were determined using the molar extinction coefficient ~280, 1.89 x 105 M-’ cm-i (20).

Sigma grade III fi-NAD and /I-NADH were used without fur- ther purification. Reduced coenzyme solutions were prepared fresh every 48 hours. Trizma base from Sigma was used for Tris buffers. The 2-(N-morpholino)-ethane sulfonic acid, A grade, was a product of Calbiochem.

Cobalt chloride and cobaltous acetate used in metal exchange studies were Baker analyzed or Fisher certified reagents. 65Zn of high specific activity (4.8 mCi per mg) was from New England Nuclear. Carrier zinc solutions and all metal standards were prepared from Fisher certified atomic absorption standards. Absolute ethanol was redistilled prior to use. All other chemicals were reagent grade.

Metal-free buffers and glassware were prepared as previously described (21), and metal-free HCl preparation was prepared by the method of Thiers (22).

Methods

Enzymatic activity of horse liver alcohol dehydrogenase was determined by the assay of Drum, Li, and Vallee (23) using a Beckman DUR spectrophotometer with a Gilford model 2000 attachment. Activity assays of yeast alcohol dehydrogenase employed a variation (NAD+ concentration, 9.2 X 10-s M) of the method of Vallee and Hoch (24).

Metal ion exchange studies were carried out by dissolving a crystalline suspension of horse liver alcohol dehydrogenase in a small volume of buffer at neutral pH and then dialyzing against a loo-fold volume of cobalt (II)-containing buffer (pH 5.5) with nitrogen flushing and continual stirring. Samples were re- moved at various times and dialyzed exhaustively at 4” with nitrogen flushing against 0.1 M Tris buffer (pH 8.0) to remove excess metals. Zinc and cobalt determinations were performed

with a Perkin-Elmer model 303 or a Jarrell-Ash Inc. (Waltham, Massachusetts) atomic absorption spectrophotometer with strip chart recorder.

Replacement by radioactive zinc of cobalt incorporated in the enzyme was carried out by a similar procedure under conditions of open dialysis. A solution of the hybrid enzyme in Tris buffer (pH 8.0) was dialyzed against a loo-fold volume of 0.1 M acetate buffer (pH 5.5) containing 65Zn(II) and carrier Zn(I1). A control solution containing only Tris buffer was also prepared. Radioactivity measurements were made with a Nuclear Chicago automatic y well scintillation counter. Enzyme and control samples contained at least lo4 cpm. The extent of 65Zn in- corporation was calculated as described previously (8).

Visible spectra of cobalt-zinc hybrids of horse liver alcohol dehydrogenase were recorded with a Cary model 15 spectro- photometer with 0 to 0.1 absorbance slide wire. Ultraviolet spectral changes produced upon inhibitor binding to these enzymes were determined with a 0 to 1.0 absorbance slide wire.

Kinetic assays of azide inhibition of horse liver aIcoho1 de- hydrogenase activity monitored the fluorescence change upon production of NADH. A Hitachi-Perkin-Elmer model MPF-2A fluorescence spectrophotometer was used to excite at 340 nm, and fluorescence emission at 460 nm was recorded. Azide inhibition of yeast alcohol dehydrogenase was determined by observing the change in absorbance at 340 nm. Azide binding to the alcohol dehydrogenases was also observed by infrared spectroscopy employing a Perkin-Elmer model 125 spectro- photometer in the manner described by Riepe and Wang (25).

Instrumentation and procedures employed in the stopped flow analyses have been described by Rudolph (26). The rates of binding of pyrazole to horse liver alcohol dehydrogenase in the presence of NAD+ were determined by monitoring the change in absorbance at 300 nm under experimental conditions de- scribed by Gilleland and Shore (16). The rates of binding of 1, lo-phenanthroline to liver alcohol dehydrogenases were determined by exciting the protein at 295 nm and following the decrease in the protein fluorescence emission, using a Corning 7-51 filter.

RESULTS

Metal Ion Exchange-Exchange of cobalt for zinc in horse liver alcohol dehydrogenase has been performed under various condi- tions of metal concentrations, anionic species, buffers, ionic strengths, and enzyme concentrations. Two rates of exchange are observed for replacement of zinc upon dialysis against 0.1 M

cobalt (II) in acetate buffer (pH 5.5) with chloride present (Fig. 1). The presence of chloride ion facilitates the metal ion ex- change.* At the lower ionic concentration and lower cobalt concentration (Fig. 1 and Table I), two rates of exchange are also apparent. Also in 0.2 M acetate buffer with no chloride present the exchange data can also be explained by two rates of exchange.

2-(N-Morpholino)-ethane sulfonic acid, which offers the advantages of being a dipolar ion and having a pK, in the region of interest (27), was also employed as a buffering agent for metal ion exchange studies. Two rates of exchange are observed (Fig. 1 and Table I), and the second order rate constants are in the same range as those observed in acetate.

The total metal atom content of the enzymes remains constant

1 David E. Drum, personal communication.

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TABLE I Rates of loss of zinc upon cobalt exchange

Exchange in acetate buffer (pH 5.5) was carried out at room

temperature and that in 2- (N-morpholino) -ethane sulfonic acid (pH 5.4) at 4“. Enzyme concentration was 5 mg per ml in ace- tate and 6 mg per ml in 2-(N-morpholino)-ethane sulfonic acid.

Apparent rate constants were calculated from the fraction of 3.7 g atoms of zinc per 80,000 g of protein not exchanged, as ob- served in Fig 1 (s).-

Experi- malt co++

dd

1 0.10 2 0.10 3 0.05 4 0.10

Buffer e&-

‘u

0.10 M Acetate 0.10 0.025 M Acetate 0.17 0.012 M Acetate 0.09

0.02~2-N-Morpho- 0.20 lino-ethane sul- fonic acid

TABLE II

r

0.3 0.3 0.15 0.3

k1 ka /

0.58 2.0 21.1 1.4

0.82 1.6

20.58 0.8

Metal content and enzymatic activity of horse liver alcohol

dehydrogenase upon zinc exchange with cobalt

Conditions were as described in Table I, Experiment 3.

Time of exchange Zinc Cobalt (Zinc +

Cobalt) SpdiC

activity

hrs I g a4oms/80,000 g protein I

0 3.7 3.7 13.2

8 1.6 2.1 3.7 8.5

16 1.4 2.15 3.6 8.4

24 1.3 2.45 3.8 7.9

32 1.16 2.5~ 3.7 i.5

after exchange and then dialysis against metal free buffers to remove extraneous metal ions, as shown in Table II. The enzymatic activity changes significantly upon replacement of the two easily exchangeable metals by cobalt and only slightly thereafter through about 40 hours of exchange. For longer periods of exchange the fraction of zinc not exchanged still fol- lowed the observed rates closely, but longer periods of time were necessary to remove all extraneous metals, and a further decrease in enzymatic activity resulted.

Exchange studies with 65Zn(II) have shown that replacement of zinc in the native enzyme by cobalt is specific. A hybrid enzyme containing a fraction of nonexchanged native zinc and a fraction of cobalt was analyzed for total metal atom content. This hybrid enzyme was then dialyzed against a 65Zn(II) con- taining acetate buffer under conditions described in Table III.

Samples of the enzyme solution were analyzed for amounts of 66Zn incorporated. The enzyme solution was then dialyzed against metal-free 0.1 M Tris buffer (pH 8.0) and analyzed for total zinc and cobalt content by atomic absorption. As shown in Table III, the decrease in cobalt content of the enzyme is equal to the increase in total zinc content. The total metal content (3.4 g atoms per 80,000 g of protein for this particular prepara- tion) remains constant during exchange. Furthermore, the amount of radioactive zinc incorporated agrees well with the decrease in cobalt content and the increase in total zinc content

of the enzyme. The enzyme retains its catalytic activity through this procedure.

TABLE III

65Zn replacement of cobalt in hybrid horse liver alcohol dehydrogenase

Horse liver alcohol dehydrogenase, 4.6 X 10e5 M; Zn(II), 1.5 X

lo+ M; exchange in 0.1 M acetate pH 5.5, at 4’. Aliquots were removed at various times and analyzed for 66Zn incorporation. Metal analyses reported here are those from the beginning of

66Zn exchange and after 35 hours of exchange.

g atoms/8?,000 g protezn

g atoms@,000 g protezn

g atoms/8(),000 g )rotern

1.55 1.8 0 3.4

2.8 0.6 1.4 3.4

i

,060

Specific activity

6.5 6.7

00 0 2.50 mM A 5.63 mM A 0.33 mM l 12.50 mM

1 2 3 4 5

l/[ETHANOLI mM-’

FIG. 2. Inhibition of horse liver alcohol dehydrogenase by sodium aside. 5.1 X UP2 M enzyme, 6.9 X 10-d M NAD+ in 0.1 M phosphate buffer, pH 8.5. The initial rate of change of fluores- cence emission at 460 nm (Afluor chart divisions per unit time.

(60 ,,I is presented in arbitrary Inhibition data were fit by a hy-

perbolic least squares program and then presented as double reciprocal plots. The insert shows a weighted least squares linear replot of slopes and intercepts from the double reciprocal plots.

Kinetic Studies of A.&de Inhibition-Kinetic results of azide inhibition of native horse liver alcohol dehydrogenase are presented according to Lineweaver and Burk (28) in Fig. 2. These data show that azide is kinetically competitive with the substrate ethanol. The inhibitor constant, Ki, under the conditions described, is 1.60 f 0.22 mM. For a hybrid enzyme containing 2.0 g atoms of cobalt and 1.6 g atoms of zinc per

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2818 Binding of Competitive Inhibitors to Alcohol Dehydrogenases Vol. 246, No. 9

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FIG. 3. Difference infrared spectrum of azide binding to yeast alcohol dehydrogenase in the presence of coenzyme. 5.6 X 10e4 M enzyme, 5.7 X 1OP M NAD+, and 2.0 X 1OW M azide. The peak at 2070 cm-l represents azide bound to the enzyme-coenzyme complex, and the peak at 2048 cm-1 represents free azide in solu- tion. The gas phase infrared spectrum of deuterium chloride was superimposed on that of the aqueous enzyme solution for calibra- tion (see Reference 30).

20165 20f8

F R E 11 U E N C Y (cm-‘) FIG. 4. Difference infrared spectra of azide binding to horse

liver alcohol dehydrogenases. A, 3.1 X 1OP M (w/w) native enzyme, 5.9 X 1CP M NAD+, and 6.4 X 1OP M azide. The peak at 2065 cm-l represents azide bound to the enzyme-coenzyme com- plex, while the free azide has a broad absorption with a maximum at 2048 cm-‘. B, 7.7 X 1OP M NAD+, 7.4 X 10m3 M azide, and hybrid enzyme. The hybrid concentration was approximately 1 mM, which is much lower than that of the native enzyme in solution A. This enzyme contained 1.3 g atoms of zinc per 80,000 g of protein after metal ion exchange with cobalt. The observed spectrum can be fitted by the absorption peak due to free azide (2048 cm-l) and a peak with a maximum at 2064 to 2065 cm-l.

80,000 g of protein the value of Ki obtained for competitive inhibition by azide is 1.97 & 0.16 mM. Azide is also a com- petitive inhibitor of yeast alcohol dehydrogenase with an inhibitor constant, Ki, of 3.3 f 0.3 mM (determined under the activity assay conditions described above).

TABLE IV Frequencies of infrared absorption maxima of azide

and its derivatives

Compound Absorption maximum

I\:3-(aqueous)*. . Ns-Zn(II)-yeast alcohol dehydrogenase-NAD+. Ns-Zn(II)-liver alcohol dehydrogenase-NAD+. NgCo(II)-Zn(II)-liver alcohol dehydrogenase-

2049 2070 2065

NAD+ . . Nr-Zn(II)-diethylenetriaminea. N3-Co(II)-diethylenetriamines Nz-Zn(II)-carbonic anhydrasee.. Na-Co(II)-carbonic anhydrasea.. CH3Nsh.

2064-2065 2085 2067 2094 2085 2143

a Riepe and Wang (25). b Eyster and Gillette (29).

Spectroscopic Studies of Azide Binding-To gain further in- formation about the nature of azide binding, the infrared spectra of azide in the presence of NAD+ and alcohol dehydrogenases were observed. In addition to the absorption peak of free azide with a maximum at 2048 cm+, a peak is observed at 2070 cm-1 in the presence of yeast alcohol dehydrogenase and NADf (Fig. 3 and Table IV). Only the absorption peak of free azide was observed with solutions of coenzyme and aside, coenzyme and azide in the presence of an inert protein, or yeast alcohol dehydrogenase and azide in the absence of coenzyme. Satura- tion of the absorption peak at 2070 cm-l occurred after addition of 4 equivalents of azide per mole of enzyme. The absorbance of the bound azide peak decreased upon addition of ethanol to the mixture.

The infrared spectrum of a solution of horse liver alcohol dehydrogenase, NAD+, and azide exhibits an absorption peak at 2065 cm-’ in addition to the absorption peak of free azide (Fig. 4A and Table IV). Only the absorption peak of free azide was observed in the spectrum of a solution containing the liver enzyme and azide but lacking coenzyme.

The increase in the stretching frequency upon binding to the zinc alcohol dehydrogenases (Table IV) compared with free azide in solution is in the same direction as that observed upon binding to zinc model complexes and in the studies of binding to the metal in carbonic anhydrase (25). However, the magnitude of the observed shift in alcohol dehydrogenase solutions is much smaller.

To further investigate the nature of azide binding, the infrared spectrum of azide in a solution of the hybrid horse liver alcohol dehydrogenase and NADf (Fig. 4B) was analyzed. In addition to the absorption of free azide at 2048 cm-l, a shoulder is ob- served in the region characteristic of azide bound to the enzyme (2060 to 2070 cm-‘). By subtracting the absorption due to free azide, the experimentally observed spectrum can be es- plained by the absorption of free azide (2048 cm-‘) and azide bound to the enzyme (maximum at 2064 to 2065 cm-l). The observed stretching frequency of azide bound to cobalt model complexes is lower than that of azide bound to zinc model complexes; similarly, the frequency of azide bound to cobalt in carbonic anhydrase is lower than that of azide bound to zinc in carbonic anhydrase (Table IV) (25). An absorption peak at 2064 to 2065 cm-l due to aside bound to the hybrid enzyme,

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Issue of May 10, 1971 J. M. Young and J. H. Wang 2819

78b bb@

FIG. 5. Difference visible snectrum of a hvbrid horse liver alcohol dehydrogenase. Enzyme concentration2 was 4.0 X 1W5 M. The spectra of solutions in a 30-mm path length cell are normalized to 800 nm. The lower line is the difference spectrum of 0.1 M Tris buffer (pH 7.5) dialyzate. The metal content was 1.6 g atoms zinc per mole of enzyme and 1.8 g atoms of cobalt per mole of enzyme.

compared with the absorption peak at 2065 cm-l due to azide bound to the zinc enzyme, does not support the possibility that azide binds to cobalt in the hybrid liver alcohol dehydrogenase.

The visible spectrum of a zinc and cobalt hybrid enzyme (Fig. 5) provides another characteristic feature by which to examine inhibitor binding. The observed maxima at 655 nm and 740 nm are at the same wave lengths as those first rcportcd for the totally cobalt-substituted enzyme (2). The molar extinction coefficients, for a hybrid enzyme containing 1.8 g atoms of cobalt and 1.6 g atoms of zinc, are e6s5 = 590 M-’ cm‘-’ and e740 = 330 M-l cm-1.2 The absorption maxima in the visible spectrum of the hybrid enzyme occur at the same wave lengths in solutions of pH 7.0, 8.4, or 9.1 (0.1 M phosphate buffer). No change in the wave lengths of the absorption maxima is observed upon addition of NAD+.

Solutions of 3 to 4 X 10-j M hybrid enzyme2 and -1 X lOA M

NAD+ in 0.1 M phosphate buffer (pH 7.0 or 8.4) were prepared to give an absorbance of about 0.06 at 650 nm in a cell of 30-mm path length. Sodium azide was added in increments to a final concentration greater than 10-l M. The visible difference spectra showed no change in the regions of the absorption maxima upon addition of azide.

Pyraxole Binding-Formation of a tightly bound liver alcohol dehydrogenase-NAD+-pyrazole complex was demonstrated by the differential absorption spectral studies of Theorell and Yonetani (31). Similar changes in the ultraviolet absorption spectrum are observed upon addition of NAD+ and pyrazole to a solution of hybrid alcohol dehydrogenase. The visible spectra of these solutions were also examined. For example, the spectrum of a solution of hybrid enzyme (8 X lo+ M) and NAD+ (9 X lop4 M) in 0.1 M Tris buffer (pH 7.5) in a cell of 50-mm path length was recorded. Upon addition of pyrasole

2 The molar extinction coefficients are calculated from the enzyme concentration as determined by the absorbance at 280 nm and the specific absorptivity for the native enzyme. The e2*0 for the hybrid enzyme may differ somewhat from that of the native enzyme (see Reference 1).

.’ 0.8-

‘;; 0.6- % - ///lj . 7 Se 0.4-

/

l . /’ 0.2- ;/*

/’ - I

, i io io 3b ;0

[PYRAZOLE]-’ ,M-’

FIG. 6. Rates of binding of pyrazole to horse liver alcohol dehydrogenase in the presence of NAD+. Experimental con- ditions were as described in Table V. Apparent first order rate constants were determined from stopped flow analysis of the change in absorbance at 300 nm (16).

TABLE: V Binding of pyrazole and l,lO-phenanthroline to

alcohol dehydrogenases

Complex 1 k

Zn(II)-liver alcohol dehydrogenase-NAD+-pyr- zolea...........................................

Co (II) -Zn(II) -liver alcohol dehydrogenase-NAD+- pyrazoleG......................................

Zn(II)-liver alcohol dehydrogenase-NAD+-pyr- azoleb . . .

Zn(II)-liver alcohol dehydrogenase-l,lO-phenan- throlinec.......................................

Co (II) -Zn (II) -liver alcohol dehydrogenase-1 , IO- phenanthroline”

‘k-1 ser x 10-4

1.4

3.3

2.8

6d

12

a Pyrazole binding in 0.1 M Tris buffer, pH 7.5, used final con- centrations of 4 X 10-S M NAD+, and either 9 X 1OM6 M native liver alcohol dehydrogenase or 8 X low6 M hybrid liver alcohol dehydrogenase.

b Pyrazole binding in 0.1 M phosphate buffer, pH 7.0, was carried out with final concentrations of 4 X 10ea M NAD+ and 6 X 10MB M native liver alcohol dehydrogenase.

c For 1, lo-phenanthroline binding the final concentration of native liver alcohol dehydrogenase was 2.2 X lOWe M and that of hybrid enzyme was 4.8 X 10~6M. 1, IO-Phenanthroline concentra- tion was varied from 2 X 10e6 M to 3 X lo-* M.

d Shore (32) has mentioned that the rate constant obtained by following the change in absorbance at 297 nm is equal to 9 X 103 M-’ set-I.

to the solution, no change was observed in the region of absorp- tion maxima of the visible spectrum.

Stopped flow kinetics has been employed to compare the rates of binding of pyrazole with native and hybrid liver alcohol dehydrogenases. Our observations for pyrazole binding to the native enzyme in 0.1 M phosphate buffer (pH 7.0) are presented in Fig. 6. For pyrazole concentrations ranging from 6 mM+ to

38 rnM-l the rate of change of k-l is linear. These values of lc-1 differ by an order of magnitude from those of Gilleland and

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Binding of Conapetitive Inhibitors to Alcohol Dehydrogenasev Vol. 246, No. 9

t+-8OOmsec+i FIG. 7. Oscilloscope tracing of t,he time course of decrease in

fluorescence emission of native horse liver alcohol dehydrogenase upon binding of 1, IO-phenanthroline. Experimental conditions were as described in Table V. The lower line corresponds to completion of reaction, t,. The apparent first order rate constant, kr = 1.53 set-1 and the half-time, t+ = 0.45 sec.

Shore (16). At, high pyrazole concentjrations the binding still follows apparent first order kinetics, but the rate of change of k-l is no longer linear.

The bimolecular rate constant determined from data in the

linear portion of Fig. 6 is presented in Table V. Binding of

pyrazole to hybrid alcohol dehydrogenase also proceeds with apparent first order kinetics. Significantly, the calculated second order rate constant for binding of pyrazole to the hybrid enzyme is twice that calculated for the native enzyme (Table V).

1 ,I@Yhenanthroline Bin&g-Characteristic ultraviolet spec- tral changes are observed upon binding of 1, lo-phenanthroline to zinc horse liver alcohol dehydrogenase (10, 13, 15), and a change also results upon binding to hybrid enzyme. However, binding of this coenzyme-competitive inhibitor to the hybrid enzyme reported above results in no observed change in the region of absorption maxima of the visible spectrum.

A typical oscilloscope tracing shows a decrease in protein fluorescence emission as a function of time after mixing 1 ,lO- phenanthroline and the alcohol dehydrogenase from horse liver (Fig. 7). The result of a plot of log (Rt - R,) versus time where Rt and R, are the fluorescence intensities at time, I, and infinite time, respectively, can be fitted by a straight line. This shows that the binding follows pseudo-first order kinetics.

DISCUSSION

These studies distinguish two classes of metal binding sites in horse liver alcohol dehydrogenase by the observed rates of cobalt substitution of native zinc. The specificity of the replacement by cobalt of the two “easily exchangeable” zinc atoms is demonstrated by the reverse 65Zn(II) exchange studies.

Furthermore, only 2.0 g atoms of zinc per mole of enzyme exchange with 6jZn(II) in acetate buffer under the conditions reported by Drum et al. (8). The exchange of cobalt in the hybrid enzyme with 65Zn(II) in acetate buffer than seems to indicate that those zinc atoms which are “easily exchangeable” with Co(II) in acetate buffer belong to that particular class which exchange with Zn(II) in acetate buffer.

The implication from the observed rates of exchange (Fig. 1) and the constant value of the total metal atom content of the enzymes (Table II) is that the replacement of all metal atoms in the enzyme by cobalt is specific. This is substantiated by the report of Drum and Vallee (1) of the preparation of a totally cobalt-substituted aIcoho1 dehydrogenase from horse liver by exchange of the totally 6”Zn(II)-labeled enzyme with cobalt in acetate buffer.

A crude comparison of the rates of replacement of the metal atoms in liver alcohol dehydrogenase by zinc and cobalt can be made, if the results of Drum et al. (8) are considered in terms of total zinc atom content of the enzyme rather than only 2.0 g atoms of zinc per mole of enzyme. Although the rates of exchange are critically dependent on many variables, in 0.1 M

acetate buffer (pH 5.5) and approximately 7 x 10e5 RI alcohol dehydrogenase, the bimolecular rate constant, ki, for zinc ex- change is roughly 1000 times greater than that for cobalt ex- change of the “easily exchangeable” metal atoms. This differ- ence should be even greater for a more valid comparison in which the experiments are performed at the same temperature.

A comparison of inhibitor binding to hybrid and zinc alcohol dehydrogenases can provide valuable information about the active sites of the enzymes. The infrared absorption studies of azide binding to native zinc alcohol dehydrogenases in the presence of NADf are consistent with azide binding to zinc metal ions in the proteins, but they are certainly not definitive. The infrared spectrum of azide in a solution of hybrid liver alcohol dehydrogenase, however, gives no evidence of binding of azide to cobalt in this enzyme. In none of these spectra is the observed asymmetric stretching frequency in the region char- acteristic of carbon-azide bonds (e.g. CH3N3), which shows that azide does not form an addition compound with the oxidized nicotinamide moiety upon binding to alcohol dehydrogenases.

hzide inhibition of yeast alcohol dehydrogenase and both the native and hybrid horse liver enzymes is kinetically competitive with substrate ethanol. The calculated azide inhibitor con- stants for zinc and hybrid liver enzymes are equal within esperi- mental error. Furthermore, no change in the visible spectrum of the hybrid enzyme was observed upon binding of the oxidized coenzyme or upon addition of azide to the enzyme-coenzyme complex under the various conditions employed. These results, therefore, indicate that azide probably does not bind to cobalt in the hydrid enzyme. Azide could bind to another cationic group, either alone or including coordination to zinc, or form a molecular complex with enzyme and coenzyme.

13inding of pgrazole, a neutral inhibitor under the conditions esamined, to the hybrid enzyme in the presence of coenzyme is verified by a change in the ultraviolet spectrum. However, no change in the visible spectrum is observed, and a change would be expected upon binding of pyrazole to cobalt. Stopped flow measurements demonstrate that this binding to the hybrid enzyme, as well as to the zinc enzyme, follows pseudo-first, order kinetics. The calculated second order rate constants show that binding to the cobalt-zinc hybrid enzyme is at least as fast as, and perhaps faster by a factor of 2 than, binding to the native enzyme. It is difficult to accept these rates as indicating binding of pyrazole to cobalt and zinc, respectively, as discussed below.

l,lO-Phenanthroline, a strong field ligand, also produces a change in the ultraviolet spectrum but no change in the visible spectrum upon binding to the hybrid enzyme. Again, stopped flow studies show the rate of binding to the hybrid enzyme to be

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Issue of May 10, 1971 J. M. Young and J. H. Wang

at least as fast as, if not faster by a factor of 2 than, binding to the native zinc alcohol dehydrogenase.

In solution the rates of binding of bidentate ligands to cobalt is slower than to zinc. In particular, Holyer et al. (33) reported the bimolecular rate constant for binding of 1 , 10.phenanthroline cobalt to be -1.5 X lo5 M-I se@, while that for binding to zinc is 23.0 X lo6 M-l set?. These rate constants are orders of magnitude greater than those for 1, lo-phenanthroline binding to alcohol dehydrogenases. If we assume that the cobalt metal ions in the hybrid enzyme have the same geometry as the zinc metal ions they replaced in the native enzyme, then we cannot interpret the observed rates to indicate that the rate-limiting step in the binding of 1, lo-phenanthroline to the hybrid and zinc enzymes is the displacement of a water molecule from the inner hydration sphere of cobalt and zinc, respectively.

These results may be interpreted to indicate that all of the inhibitors investigated bind to sites separate from the two metal ions in horse liver alcohol dehydrogenase which are “easily exchangeable” with cobalt in acetate buffer. Although kinetic competition does not dictate that inhibitor and substrate (or coenzyme) bind at precisely the same site on the enzyme surface, we may infer that the substrate probably also binds to a site separate from these “easily exchangeable” metal ions. Prelimi- nary x-ray crystallographic results also suggest that these metal atoms may be located far from the coenzyme binding site (34).

We must again ask the question, which particular class of metal ions in horse liver alcohol dehydrogenase exchanges rapidly with cobalt in acetate buffer? As discussed above, the observation that cobalt in the hybrid enzyme can be replaced by exchange with @Zn(II) in acetate buffer suggests that the metal ions which are “easily exchangeable” with cobalt in acetate belong to that particular class which exchanges with @Zn(II) in acetate buffer (8). Since loss of enzymatic activity correlates closely with the removal of these zinc ions (8, 9), and since the characteristic interactions with such inhibitors as 1, IO-phenanthroline are abolished upon their removal (lo), it has been proposed that this particular class of metal ions plays a role in the catalytic process. If we accept that the metal ions which are easily exchangeable with Co(I1) in acetate buffer are the same as those which exchange with e5Zn(II) in acetate, then the studies presented here indicate that these metal ions in horse liver alcohol dehydrogenase do not bind inhibitors and may in fact have no direct catalytic function. Instead, these metal ions may be present simply to maintain the three-dimensional structure of the enzyme. On the other hand, no conclusion can be drawn from these studies concerning the roles of the more slowly exchanging zinc ions in horse liver alcohol dehydrogenase.

Acknowledgments-We wish to express particular gratitude to Professor Joseph E. Coleman for use of the Cary 15 and Jarrell-Ash atomic absorption spectrophotometers. We thank Professor Julian M. Sturtevant and Dr. Stephen A. Rudolph for use of the stopped flow apparatus as well as suggestions about the fluorescence studies, and Professor Lubert Stryer for use of the y well scintillation counter. Kinetic data were analyzed by programs provided by Dr. H. W. Duckworth.

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J. Maitland Young and Jui H. WangThe Nature of Binding of Competitive Inhibitors to Alcohol Dehydrogenases

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