[en] Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Hernandez Valladares, M.
Felici, A.
Weber, Georges ; Université de Liège - ULiège > Département de physique > Physique nucléaire, atomique et spectroscopie
Adolph, H. W.
Zeppezauer, M.
Rossolini, G. M.
Amicosante, G.
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Zn(Ii) Dependence of the Aeromonas Hydrophila Ae036 Metallo-Beta-Lactamase Activity and Stability
Publication date :
23 September 1997
Journal title :
Biochemistry
ISSN :
0006-2960
eISSN :
1520-4995
Publisher :
American Chemical Society, Washington, United States - District of Columbia
De Meester, F., Joris, B., Reckinger, G., Bellefroid-Bourgignon, C., Frère, J.M., Waley, S.G., (1987) Biochem. Pharmacol., 36, pp. 2393-2403
Felici, A., Amicosante, G., (1995) Antimicrob. Agents Chemother., 39, pp. 192-199
Felici, A., Amicosante, G., Oratore, A., Strom, R., Ledent, P., Joris, B., Fanuel, L., Frère, J.M., (1993) Biochem. J., 291, pp. 151-155
Frère, J.M., (1995) Mol. Microbiol., 16, pp. 385-395
Galleni, M., Amicosante, G., Frère, J.M., (1988) Biochem. J., 255, pp. 123-129
Gomez-Ortiz, M., Gomis-Rüth, F.X., Huber, R., Avilés, F.X., (1997) FEBS Lett., 400, pp. 336-340
Hernandez Valladares, M., Galleni, M., Frère, J.M., Felici, A., Perilli, M., Franceschini, N., Rossolini, G.M., Amicosante, G., (1996) Microb. Drug. Resist., 2, pp. 253-256
Holmquist, B., Vallee, B.L., (1974) J. Biol. Chem., 249, p. 4601
Jackson, M.J., Zinc in Human Biology (1989) Human Nutrition Reviews. Physiology of Zinc: General Aspects, pp. 1-10. , Mills, C. F., Ed. Spring-Verlag, Heidelberg
Johansson, S.A.E., Campbell, J.L., (1988) PIXE: A Novel Technique for Elemental Analysis, , Wiley, New York
Larsen, K.S., Auld, D.S., (1989) Biochemistry, 28, p. 9620
Larsen, K.S., Auld, D.S., (1991) Biochemistry, 30, p. 2613
Massidda, O., Rossolini, G.M., Satta, G., (1991) J. Bacteriol., 173, pp. 4611-4617
Payne, D.J., Cramp, R., Bateson, J.H., Neale, J., Knowles, D., (1994) Antimicrob. Agents Chemother., 38, pp. 991-996
Robaye, G., Weber, G., Delbrouck, J.M., Roelandts, I., Bartsch, P., Collignon, A., (1981) Nucl. Instrum. Methods, 181, pp. 59-62
Segatore, B., Massidda, O., Satta, G., Setacci, D., Amicosante, G., (1993) Antimicrob. Agents Chemother., 37, pp. 1324-1328