The molecular kinetics of the urea-urease system. VI. The activation of urease by amino acids

The molecular kinetics of the urea-urease system. VI. The activation of urease by amino acids

The Molecular Kinetics of the Urea-Urease System. VI. The Activation of Urease by Amino Acids Mary Colman Wall and Keith J. Laidler From the Departmen...

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The Molecular Kinetics of the Urea-Urease System. VI. The Activation of Urease by Amino Acids Mary Colman Wall and Keith J. Laidler From the Department

of Chemistry,

The Catholic University Washington, D. C.

of America,

Received August 11, 1952 INTRODUCTION

A number of years ago Kato [(2-5) ; see also (1, 7)] published the results of extensive investigations on the effect of glycine on the ureasecatalyzed hydrolysis of urea. His work was done with a crude urease extract, and he used a phosphate buffer. The curve of rate versus substrate concentration passes through a maximum when this buffer is employed, and Kato reported that the addition of sufficient glycine increased the rate to the right of the maximum, so that the curve then more closely resembled a typical Michaelis curve. The results of the present investigation, however, do not confirm this result, but indicate that the situation is in reality more complicated. The reason for the discrepancy between Kato’s results and ours is considered later. The object of the work reported here was to investigate the influence of amino acids on the rate of the urease-catalyzed hydrolysis of urea, at various substrate concentrations and using the phosphate buffer and also the inert trishydroxymethylaminomethane (THMAM)-Ha04 buffer. RESULTS

The experimental procedure was exactly as described in part IV of this series (9). The first investigation involved measuring rates at a series of substrate concentrations in the presence of 0.026 M (0.2$!$) glycine, and this was done in THMAM-H2S04 (pH 7.13) and phosphate (pH 6.60) buffers. The results are shown in Figs. 1 and 2. Two sets of runs using nn-alanine and L-tyrosine were also made in both buffers for urea concentrations of 0.005 and 0.83 M: one set of runs was made in solutions made 0.026 312

THMAM-H2SOs pH 7.13


















FIG. 1. Plots of initial rate (moles/l./sec.) H&O1 buffer at pH 7.13; 20.8%. Phosphate RH 6.60

5 x >




vs. concentration of urea; THMAM-








M in alanine, and the second in solutions saturated with tyrosme (0.00226 M). The rates are listed in Tables I and II. The dependence of the activation effect on the concentration of added glycine was studied by measuring rates in solutions of 0.83 M urea in TABLE Initial


Rates in Presence and Absence of Amino


(0.83 M urea; 2W3”C.) Initial BufIers


rate X 106

Noaydinino 0.0026 M

0.026 M oh&mine


0. 0226 M btyrosine


Phosphate. ................ THMAM-HsSOa ...........

6.60 7.13

4.3 6.2

10.9 12.5






8.8 -

TABLE Initial


Rates in Presence and Absence of Amino


(0.005 M urea; 20.8”C.) I



rate X 10’

,Phosphate................ THMAM-H

*SO1. ......... TABLE


of Glycine (0.83 M urea; phosphate buffer at pH 6.60; 20.8’C.) Initial

Rates for Various Concentrations

Glycine concentration moles/l.

0.000 0.0026 * 0.0065 0.0260

Initial rates X 10’ mdesjl. /Sec.

4.3 8.5 9.1 9.9

phosphate buffer at pH 6.60. The glycine concentrations (molar) selected were: 0.0026, 0.0065, 0.026 (0.2%), 0.26, and 0.83. The rates for the first three concentrations are given in Table III; no reliable results could be obtained for the two highest concentrations with the procedure





used since under these conditions reaction between glycine and the Nessler reagent was appreciable. DISCUSSION

Throughout this section the following notation will be used. R represents the ratio of rates in the presence, and in the absence, of amino acid. [S] represents the concentration of substrate (urea) in moles per liter, and [S]* represents the urea concentration corresponding to the maximum rate in the absence of amino acid; [S]* is 0.15 M for both buffers. TABLE Approxima.te


R Values at Various (Temp.

Urea Concentrations

= 20.8”C.) R


concentration ~#k$



0.005 0.025 0.150 = Ly* 0.830

1.4 1.4 1.4 2.5

1.4 1.4 1.4 2.0

The main results obtained in connection with the effect of amino acids may be summarized as follows: 1. The rate is increased for all [S] ‘in both buffers. 2. For both buffers the value of R is independent of [S] for S Q [Sl* (see Table IV). 3. For both buffers R is considerably greater at high [S] than at values less than or equal to [S]*, as can be noted in Table IV. 4. Increases in rate are of the same order of magnitude in 0.026 M alanine as in 0.026 M (0.2oJ,) glycine. A somewhat smaller value of R is found for the solution saturated with tyrosine; this is probably due to the lower concentration. Kato reported that when solutions were made 0.27c in giycine the rates at high substrate concentrations were increased approximately to the maximum rate, but that rates at [S] < [S]* were unaffected; i.e., the only effect is that of removal of substrate inhibition. The results of the present work are seen to be quite different from this; in both buffers there is activation at all substrate concentrations, the effect being greater







the greater the substrate concentrations.Table IV shows that for [S] < [Xl* the activation effect is independent of [S] and of the nature of the buffer, but that at [S] > [S]* it is different for the two buffers. In discussing the mechanism of the activation by amino acids it is as well at the outset to exclude one possibility: the fact that the results are similar in the two buffers excludes the hypothesis that the amino acids in some way counteract inhibition due to sodium and potassium ions present in the buffer. (a) A simple hypothesis would be that some of the urease molecules are present in solution in the form of a less active form E, and that by interaction with amino acid they are converted into a more active form E*

If this occurs in a noncompetitive fashion (i.e., if the amino acid and the substrate interact with the enzyme at different sites), it would imply the same degree of activation at all substrate concentrations. This hypothesis is entirely consistent with the data for [S] < [S]*, since here the activation is independent of [S]; some additional mechanism is, however, required for [S] > [S]*, where the activation is greater. (b) Since at high substrate concentrations substrate inhibition is important, the simplest hypothesis would seem to be that the amino acids in some way reduce the extent of this inhibition. This possibility may be considered further with reference to the hypothesis of substrate inhibition suggested in part I (6), according to which the enzyme has two neighboring active sites on one (&) of which (for reaction to occur) a urea molecule must be adsorbed, on the other (8~) of which a water molecule must be adsorbed (i.e., a urea molecule must not be adsorbed). A simple way in which amino acid could reduce substrate inhibition is for it to become adsorbed in some position on the enzyme where it can sterically’prevent adsorption of urea on &, but where it does not prevent adsorption of water on X2; this is clearly quite plausible in view of the much larger size of urea as compared with water. This hypothesis gains some support from the fact that the activating effect is greater in phosphate buffer than in THMAM-HzSOd (cf. Table IV), since in the latter buffer the extent of inhibition by urea is less than in the former. (At 0.83 M urea concentration the rate is reduced to 0.59 of the maximum in THMAM and to 0.45 in phosphate.) However the hypothesis is not alone capable of explaining the extensive activation





found at the lowest urea concentrations, since here the amount of substrate inhibition is negligible. This effect must be explained by hypothesis (a). It seems necessary to conclude, therefore, that two separate effects are involved: the activation of the enzyme in a noncompetitive fashion and the removal of substrate inhibition. A simple formulation which covers both effects is as follows. It is assumed that the enzyme is normally present in a form E, and that this is converted into a more active form E* by the action of the amino acid



Both E and E* form complexes of the usual type with substrate; and these give rise to products. E+A

G X + products

E* + S ti X* -+ products

(2) (3)

To account for substrate inhibition it is postulated that both X and X* combine with additional substrate to form inactive complexes: x+sey




The amino acid, furthermore, combats substrate inhibition by combining with X and X* to give complexes that will still react to form products : X + A * 2 -+ products


X* + A ti Z* -+ products


Application of the steady-state treatment to this set of reactions gives rise to a very clumsy kinetic expression, but one which satisfactorily accounts for the behavior. In the light of the above discussion it is possible to suggest an explanation for the somewhat different behavior found by Kato, according to whom there is no activation for [S] < [S] * and who found for [S] > [S] * activation up to the maximum rate observed in the absence of glycine. Kato was working with a crude extract that contained proteins other than urease, and possibly amino acids also. Proteins, as well as amino acids, are known to activate urease (8), and it is possible that in Kato’s






system they were present in sufficient amount to bring about maximum activation according to mechanism (1) above; i.e., E was entirely converted into E*. There would therefore be no activation for [S] < IS]*, where mechanism (1) predominates. There could still be activation at higher concentrations, however, since here mechanism (2) predominates. SUMMARY

The activating effects of glycine, nn-alanine and n-tyrosine on the urea-urease system have been investigated over a range of substrate concentrations and in two buffer systems: phosphate and trishydroxymethylaminomethane-H&Sod. It is found that at a substrate concentration lower than that which corresponds to the maximum rate (in the absence of activator) the effect is independent of substrate concentration. At higher substrate concentrations, where substrate inhibition is important, an additional activation is observed. The results are explained in terms of the hypothesis that two separate effects are involved: (a) a general noncompetitive activation, which would give rise to the same degree of activation at all substrate concentrations, and (b) a reduction of substrate inhibition, which would predominate at high substrate concentrations.

1. 2. 3. 4.

5. 6. 7. 8. 9.


W. J., J. Am. Chem. Sot. 49, 3199 (1926). N., J. Pharm. Sot. Japan 498, 867 (1922). N., J. Pharm. Sot. Japan 494, 228 (1923). N., Biochem. 2. 136, 498 (1923). KATO, N., Biochem. 2. 139, 352 (1923). LAIDLER, K. J., AND HOARE, J. P., J. Am. Chem. Sot. 71,2699 (1949). ROCKWOOD, E. W., AND HUSA, W. J., J. Am. Chem. Sot. 46, 2678 (1923). SUMNER, J. B., J. Biol. Chem. 76, 149 (1928). WALL, M. C., AND LAIDLER, K. J., Arch. Biochem. and Biophys., 43,299 (1963).