and 76.39 for K+, N a + and C1-, respectively; the values obtained from the extrapolated transference datal0are 73.50, 50.10 and 76.35.
relatively small difference between .ioand A:, with a resulting limiting Kohlrausch ratio not cliff ering greatly from unity. Thus to attain as high a numerical precision as possible in the ionic quantities, 0.810 I leading and indicator ions should have as large :i difference in ionic conductance as possible, and this should be borne in mind in selecting the electrolytes for study in any new solvent. I t should also be noted that while the extrapolation procedure outlined above is probably adequate 0.800 for strong electrolytes, the extrapolation, if ion pair 0 0.01 0.02 formation is appreciable, would probably require c. the use of ionic rather than stoichiometric concenFig. 1.-Thc qiiaitity 1'" AS a function of KCI coticcritra- trations in eq. 6 and in the analog of eq. 7. We tion; the radii of the circles correspond to the apparent believe, however, that these results show that liinitprecision of the measurements. ing ionic conductances can be determined with reaThe results obtained here €or the liiniting tram- sonable precision for any solvent in which precise ference numbers and ionic conductances illustrate conductance measurements are possible. In conclusion, we wish to express our thanks to one restriction on this method. Although the extrapolated Y O differs from that computed from the hlr. R. H. Chappell for constructing the conductlimiting transference data by no more than the ance cells, and to the National Research Council of numerical precision of the latter (a part in 4000) Canada for a grant in aid of this research and for the ionic conductances deviate by a part in 1800 for the award to D.R.M. of a studentship and a fellowthe faster ions, and by a part in 1000 for the slow- ship. est. Inspection of eq. 5 shows that this is due to the TORONTO, O S T A R I O , CANADA
r
t l
[CONTRIDUTIOS FROM THE DEPAR.TMEMT OF
CHEMISTRY, THEUNIVERSITY
OF
TEXAS]
The Inhibition of Urease by Various Metal Ions BY
1 ~ 1 L L 1 . 4 XH.
R.
SHA\\V
RECEIVED J U N E 17, 1953 Data 011 the relative toxicity of metal ions toward the enzyme urease have been collected from the literature. I t has been found possible to arrange the common metal ions in a toxicity sequence. Correlation of toxicity with various properties of the metal ions is discussed and illustrated on the basis of a model mechanism.
Introduction The enzyme urease is highly sensitive to trace quantities of metal ions. Different metals exhibit quite different behavior in their ability to act as enzyme inhibitors. I n the case of urease, for example, the silver ion1V2is an extremely efficient inhibitor, while the manganous ion is relatively very weak.3 A search of the literature has revealed that enough data are now available to order the common metal ions in a tentative sequence of relative inhibitory efficiency. It is the purpose of this investigation: to summarize the available data in such a sequence; to define a quantitative functional measure of inhibitory efficiency; and to correlate this quantity, if possible, with soine fundamental propcrty of the metals. Mathematical Development I t was demonstrated in a previous communication4 that the inhibition index, for a first-ordrr Michaelis-Mcnten
system, was given by the equation ___ ~
( I ) J . B. Surnner and K , LIyrback, %. fihysioi. C'ilcm., 189, 218 (1930). ( 2 ) J . F. Ambrose, G. B. Kistiakowaky a n d A. G . Kridl, THIS J O U R N A L , 73, 1232 (1951). (3) A . L. Dounce. National Nuclear Energy Series, Div. VI. Vol. 1, hIcCrarv-Hill Book Co.. I n c . , New York, N . Y . . 1010, pp. 839-848. (4) G. €3. Kistiakuwsky a n d W. H. R . Shaw, HIS J O U R N A L , 7 6 ,
GG (1Y.53).
S is the substrate concentration, Vu is the uninhibited rate of urea hydrolysis by urease and Vi is the inhibited rate, K , is Michaelis constant, K I the equilibrium constant for the combination of the free enzyme with the inhibitor, and Kz an analogous constant involving the enzyme-substrate. The concentration of inhibitor not bound to the enzyme6 is designated by "i." At an experimentally fixed substrate concentration, i t has been common practice t o determinc the concentration of inhibitor (I) necessary to produce some arbitrary inhibition ( @ A ) . Thus for all the types of inhihition listed in Table I, equation 1 may be rearranged to read PI = const. log Ki (SI
+
where Ki may be K1, Kz or K , and p refers to the ncgativc logarithm of the quantity involved. For an enzyme o h c y ing the inhibited Michaelis-Menten mechanism with the above restrictions, a comparison of PI'S for various inhihitors corresponds t o a comparison of functions that are, a t :i given temperature, linear functions of the free energy of inhibition -AF,O = RT It1 K , ( 3I If Ki = K , the total free energy of irihihitioti will be twicc that given by equation 3, since two inhihition reactions arc involved. The larger the value of +I the more efficient the inhibitor, since there is a greater loss in the free energy of ( 5 ) I n what follows i t is assumed t h a t t h e a m o u n t of inhibitor bound t o t h e enzyme is small compared t o t h e total inhibitor concentration. For inhibitor concentrations t h a t greatly exceed t h e t o t a l enzyme ciincentration, this assumption is entirely justified. For very strong iiihibitors, such as silver ion.it should be considered a s an approxiniation.
INEIIRITION OF UREASE BY VARIOUS METALTONS
April 20, 1954 TABLE I VALUESOF
THE
PARAMETERS IN EQUATION 2 FOR OUS
Type of Case inhibition
THE
VARI-
Condition
Logari t hmic term
TABLE 111 A, RELATIVE TOXICITIES OF THE VARIOUS METALIONS Investigator
TYPES OF INHIBITION Constant term
Schmidt'
DounceS
+ mK , Van de Veldeg
UncomS log Kz @+a log petitive K~ = O 3 Non-com- K2 = K I = K log K @+A petitive 4 Others K2S>>K1Km Same as case 2 Same as case 1 KmKl>> SKZ
2
Kitagawa? Best sequence
inhibition. The quantity PI is thus a quantitative measure of toxicity based on the thermodynamic consequences of the inhibited Michaelis-Menten model.
Ag Hgt+ +
Cu++
Zn++ Cd++ Pb++ co++ Ni++ Mn++
Salt used
AgN03 HgC12"
THE
Property
Using crude preparations of urease from jack bean meal, Schmidt' determined the minimum concentration of metal salt necessary t o so reduce the rate that no detectable quantity of ammonia could be found under his experimental conditions. Very dilute solutions of urea were employed (approx. 30 p.p.m.). The reaction mixtures were incubated a t 50' for five minutes in unbuffered solutions initially a t PH 7. The results obtained have been recalculated to express the inhibitor concentration in moles per liter, and the PI values derived from these calculations are recorded in Table 11. TABLE I1 PI VALUESOBTAINED BY VARIOUS INVESTIGATORS Ion
Toxicity sequence
Ag+-Hg++ > C u + + > Z n + f > C d + + > P b + + > Co++ > Ni++ > Mn++ Ag+ > H g + + > C u + + > C d + + > Co+ > M n + + Ag+, Hg++, C U + > ~ Co++ > X i + + > Cd++, Pb+, Z n + + > M n + + Hg++, C u + + > Z n + + Ag+-Hg++ > C u + + > C d + + > C o + + > N i + + > M n + + with P b + + and Z n + + u n a 4 g n e d but less than C u + +
B, OTHERPROPERTIES OF
Data
2101
Schmid to
4.73 4.7 4.7 3.8
..
2.8 2.6 2.3 0.90
.77 .04
..
*IDouncea
7.1 6 . 7 (PH 5) 5.6(pH7)
... ...
4.5
...
3.9
... 2.4
...
...
1.5 a Not completely dissociated. Unless the enzyme has equal affinity for dissociated and undissociated form, the results are somewhat ambiguous. The best recent comparative study is that of Dounce.' The observations were made a t various pH values in several buffers. Crystalline urease was used throughout: measurements were made a t 25' with a urea concentration of 3%. T o make the data comparable to those of Schmidt, the results have been converted to approximate PI values by calculating the inhibitor concentration in moles/liter necessary to produce 95% inhibition a t PH 7. I n view of the semiquantitative character of the data, the PI values reported in Table I1 should be considered as rough estimates-probably good a t best to two significant figures. Several qualitative statements concerning the relative inhibitory efficiency of metal ions can also be found in the literature. In many cases, however, it is impossible to evaluate critically the significance of these data because the original articles were not available. The results of two such researches'-O are presented in Table IIIA. (6) E. G.Schmidt, J . B i d . Chcm.. 7 8 , 53 (1928). (7) M. Kitagawa, J . Biochcm. (Japan), 10, 197 (1929); C. A . , 43, 3242 (1929). (8) A. J . J. Van de Velde, Meddel. Koninkl. Vlaom. A r a d . , 9, No. 12, 13 (1947); C . A , , 42,7803 11948). (9) I l i d . , 11, No. 1 1 (1940); C.A , , 46,9581 (1951).
METALIONS Seqnence
Insolubility of the sulfide Ag+-Hg++ > C u + + > P b + + > C d + + > Zn++ > Co++ > Ni++ > Mn++ Sum of the first and sec- H g + + > C u + + > Z n + + > C d + + > Ni++ > Co++ > hfn++ > ond ionization potentials of the gaseous atoms Pb++ H g + + > Cu++ > Xi++ > P b + + Chelate stability series > Co++ > Zn++ > C d + + > M n + + Electromotive series Ag < Hg < Cu < Pb < Ni < Co < Cd < Zn < Mn Soy bean urease preparations were used in all of these investigations. The over-all agreement of the data collected in Table IIIA is remarkably good, when one considers that enzyme preparation of various degrees of purity2 from two different sources were employed. Before turning to the correlation and interpretation of these data, the importance of the buffer in experiments of this type should be considered. Because of its excellent buffering qualities phosphate buffer has unfortunately been widely employed. Phosphate exhibits a strong complexing tendency toward metal ions.I0 Measurements made in this buffer may not reflect the true inhibitory efficiency series of the metals but only a sequence of relative complexing tendencies. As an additional complication it has been well established that phosphatellJ2 buffer interacts quite strongly with the enzyme urease. On the basis of these facts the work of M. Jacoby's has been omitted because strong phosphate buffer was employed, and the data so obtained were in conflict with results reported above.I4
Correlation Rather extensive experimental data are available supporting the contention that urease contains one or more sulfhydryl groups16@as integral parts of its catalytically active site. In view of this evidence the inhibition of urease by metal ions has been assumed to result from a reaction such as1?
(10) J. B.Sumner and K. Myrback. "The Enzymes," Vol. I, Part 1, Academic Press, Inc., New York, N. Y.,1951, p. 11. (11) K. M. Harmon and C. Niemann, J . Biol. Chcm., 177, 601 (1949). (12) G. B. Kistiakowsky, P. C. Mangelsdorf, Jr., A. J. Rosenberg and W. H. R. Shaw, THISJOURNAL, 74, 5015 (1952). (13) M.Jacoby, Biochcm. Z., 450, 211 (1933). (14) I n the work of Dounce,' measurements with the Hg + + were conducted in phosphate, but other ions were studied in maleate or utlbuffered solutions. This author recognized and emphasized the complexing tendencies of phosphate, and observed that H g + +is probably a stronger inhibitor than he reported. (15) L. Hellerman, "Cold Spring Harbor Symposium Quant. Biol.," VII, 105 (1939). (16) C. V. Smythe, J . B i d . Chem., 114, 601 (1936). (17) 1.. Massnrt, ref. 10, p. 328.
21 62
R . SFTAW
Vol. 76 TABLE 1V
pI
