The urease-catalyzed hydrolysis of urea has been found to be competitively inhibited by phosphate at pH 7.0 and 25°. The Michaelis constant of the urea-urease system has been found to be approximately 0.003 M urea and the comparable constant defining the phosphate-urease system 0.035 M phosphate

Harmon, Kent M.

Niemann, Carl

English

Caltech Authors - Main

THE COMPETITIVE INHIBITION OF THE UREASE-CATALYZED 
HYDROLYSIS OF UREA BY PHOSPHATE 
BY KENT M. HARMON* AND CARL NIEMANN 
(From the Gates and Crellin Laboratories of Chemistry,t California Institute 
of Technology, Pasadena) 
(Received for publication, August 31, 1948) 
The inhibition of the urease-catalyzed hydrolysis of urea by phosphate 
has been noted by a number of investigators (l-3), but there appears to 
be no information available regarding the nature of this inhibitory action. 
Assuming the validity of the Michaelis-Menten equation (4), it can be 
shown (5, 6) that for a system containing enzyme, substrate, and inhibitor 
; =(l+$)(+)(~)+(l+it;)(+) 0) 
where 
e = total enzyme concentration 
s = substrate concentration 
i = inhibitor “ 
P = concentration of enzyme-substrate complex 
!7 = 
‘I “ enzyme-inhibitor “ 
t = “ “ enzyme-substrate-inhibitor complex 
= observed rate for a given initial concentration of e, 8, and i 
Ik, = s(e - P - Q - d/P 
K* = i(e - p - q - r)/q 
K. = ip/r 
V = rate where i = 0 and p = e 
It follows from equation (1) that when l/u is plotted (usually as the 
ordinate) against l/s a straight line will be obtained with inhibitory action 
influencing either the slope or the ordinate intercept or both. Thus the 
type of inhibition may be defined on the basis of the effect of the inhibitory 
action upon the slope and intercept in the above plot. 
In the absence of an inhibitor (i = 0) equation (1) reduces to the Mi- 
chaelis-Menten equation 
; =(5)(;)+(+) (2) 
permitting the evaluation of P and K,. With competitive inhibition, i.e., 
when both substrate and inhibitor are presumably competing for the same 
* National Research Council Predoctoral Fellow. 
t Contribution No. 1238. 
601 
602 HYDROLYSIS OF UREA 
reactive sites, we may set K,, = 00, transforming equation (1) into 
(3) 
From equation (3) it is clear that with competitive inhibition only the slope 
will be affected, being increased by the factor (1 + (i/K,)) over that ob- 
taining when i = 0. Other types of inhibition, i.e. non-competitive, “un- 
competitive” (6), and “quadratic” (6), may be recognized in the order 
named by a proportional increase in slope and intercept (K, = K,), an 
increase in intercept with no change in slope (K, = a), and by apparently 
unrelated changes in slope and intercept (K, # K,,). 
TABLE I 
Effect of Bu$er Concentration upon Kinetics of Hydrolysis of Urea by Urease 
I 
II 
III 
- 
BUfftX 
Phosphate 
“ 
Maleate 
Glycine 
Buffer con- 
centration 
H 
0.030 
0.056 
0.109 
0.056 
0.109 
0.161 
0.267 
0.330 
0.16 
0.32 
0.53 
0.10 
0.32 
0.53 
I- 
- 
Slope, m htercept, b 
?# 
b 
___ 
8.1 1.27 6.4 
11.3 1.28 8.8 
22.1 1.26 17.5 
11.9 1.49 8.0 
19.4 1.53 12.7 
26.8 1.62 16.0 
62.0 1.39 37.6 
69.1 1.71 40.5 
7.8 1.70 4.6 
7.8 1.30 4.3 
7.8 2.30 3.4 
6.8 1.46 4.6 
7.0 1.54 4.6 
7.2 1.68 4.3 
- 
- 
KC 
0.038 
0.037 
0.034 
0.033 
0.034 
In the urease-catalyzed hydrolysis of urea in the presence of phosphate 
it has been observed (Table I, Fig. 1) that the ordinate intercept of the 
l/v versus l/s plot remains essentially unchanged with increasing phos- 
phate concentration, whereas the slope increases markedly, the increase 
being approximately linear for the lesser phosphate concentrations. Upon 
extrapolation to zero phosphate concentration a value of 4.5 was obtained 
for the slope (m), and from the slope-intercept ratio at zero phosphate 
concentration a value of 0.003 M urea was obtained for the Michaelis con- 
stant (Km) of the urea-urease system at pH 7.0 and 25”. 
For a case of competitive inhibition one may obtain from equation (3) 
the relation 
K. M. HARMON AND C. NIEMANN GO3 
m=(l++-)($) (4) 
and, using the extrapolated value of m = Km/V = 4.5, K, may be calcu- 
lated from the relation 
K, = ---5 (5) 
where i = micromoles of inhibitor per ml. For values of i varying from 
30 to 160 micromoles of phosphate per ml. an average value of K, = 0.035 
M phosphate at pH 7.0 and 25” was obtained (Table I). That the observed 
I8 .38 M 
14 
12 .287 
IO 
48 
.I81 
8 
.I09 
4 .058 
2 
FIG. 1. Effect of phosphate buffer upon the hydrolysis of urea by urease. l/u in 
(micromoles of ammonia)-1 per ml. per minute, l/s0 in (micromoles of urea)-l per ml. 
inhibition by phosphate is not simply an effect of ionic strength is shown 
by the markedly different behavior observed with maleic acid and glycine- 
carbonate buffers adjusted to pH 7.0 (Table I, Fig. 2). Increasing the 
maleic acid concentration from 0.16 to 0.53 M caused no change in slope 
and only a relatively small increase in intercept. The increase in slope 
and intercept noted with increasing concentration of the glycine-carbonate 
buffer may or may not be significant, since the observed variations are 
within the limits of experimental error. 
It is noteworthy that with the maleic acid and glycine-carbonate buffers 
a slope-intercept ratio is obtained which is in good agreement with the ratio 
of 4.5 obtained from the phosphate data upon extrapolation to zero phos- 
phate concentration, and one may conclude that the true Michaelis constant 
of the urea-urease system at pH 7.0 and 25’ is approximately 0.003 M urea. 
604 HYDROLYSIS OF UREA 
It is clear that the higher values for K,,, reported previously (7) are a con- 
sequence of the hitherto unrecognized competitive inhibitory action of 
phosphate in the urease-catalyzed hydrolysis of urea. On the basis of 
computed values for K,,, and Kc it appears that at pH 7.0 and 25” the in- 
hibitory quotient of phosphate in the urea-urease system (Kc/Km) is ap- 
proximately 12. 
Preliminary experiments with N-butylurea, N-tert-butylurea, and N- 
methylurea have indicated that these substances exert an inhibitory action 
in the urea-urease system, their effectiveness being in the order named. 
Present information does not permit definition of the nature of their in- 
+;k!i 1 
0 .04 .08 .I2 .I6 .20 
l/So 
FIG. 2. Effect of maleate and glycine-carbonate buffers upon the hydrolysis of 
urea by urease. l/u in (micromoles of ammonia)-’ per ml. per min. l/soin (micro- 
moles of urea)-l per ml. 
hibitory action, but it does appear that the action is not one of simple 
competition as was observed in the case -of phosphate. 
EXPERIMENTAL 
The procedure used for the determination of urease activity and for the 
study of the kinetics of the hydrolysis of urea by.urease at pH 7.0 and 25” 
has been described (7). In order to avoid complications arising from the 
dependence of the specific .activity of urease upon the apparent absolute 
enzyme concentration (7) -solutions of thrice recrystallized urease were 
prepared containing approximately 1 y of proteinN per ml., stabilized with 
hydrogen sulfide (7). These enzyme solutions were 0.01 M in the buffer 
and were allowed to stand at 25” for 5 hours before use. 
K. M. HARMON AND C. NIEMANN 605 
The phosphate buffers were prepared from recrystallized dipotassium 
hydrogen phosphate and potassium dihydrogen phosphate, the maleate 
buffers by the addition of solid reagent grade sodium hydroxide to a solu- 
tion of recrystallized maleic acid, and the glycine buffers by the addition 
of recrystallized sodium carbonate to a solution of recrystallized glycine. 
In every case, irrespective of the concentration of the buffer, the pH of 
the solutions was 7.0 at 25”. 
A reaction time of 3 minutes was used in all of the experiments herein 
reported. Control experiments with maleic acid buffers in which the reac- 
tion time was varied between 2 and 5 minutes showed only the usual ex- 
perimental variation. A least squares treatment, in which the data were 
weighed proportionally to the reaction velocities, was used in computing 
the slopes and intercepts of the l/v versus l/s plots. 
SUMMARY 
The urease-catalyzed hydrolysis of urea has been found to be competi- 
tively inhibited by phosphate at pH 7.0 and 25”. The Michaelis constant 
of the urea-urease system has been found to be approximately 0.003 M 
urea and the comparable constant defining the phosphate-urease system 
0.035 M phosphate. 
BIBLIOGRAPHY 
1. Van Slyke, D. D., and Zacharias, G., J. Biol. Chem., 19, 181 (1914). 
2. Krebs, H. A., and Henseleit, K., 2. physiol. Chem., 210, 33 (1932). 
3. Howell, S. F., and Sumner, J. B., J. Biol. Chem., 104, 619 (1934). 
4. Michaelis, L., and Menten, M. L., Biochem Z., 49,333 (1913). 
6. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66, 658 (1934). 
6. Ebersole, E. R., Guttentag, C., and Wilson, P. W., Arch. Biochem., 3,399 (1943-44). 
7. Peterson, J., Harmon, K. M., and Niemann, C., .I. BioE. Chem., 176, 1 (1948). 


The competitive inhibition of of the urease-catalyzed hydrolysis of urea by phosphate

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The competitive inhibition of of the urease-catalyzed hydrolysis of urea by phosphate

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