PRELIMINARY NOTES 535

PN 61053 Regulatio,1 of adenylate deaminase by adenosine triphosphate*

Brain contains an adenylate deaminase (AMP aminohydrolase, EC 3.5.4•6) tha t is ac t ivated by adenosine t r iphosphate 1-3. Part ial ly purified preparat ions of the enzyme have been obtained from acetone powder extracts of ox brain 2 and dog brain 3. Bo th types of preparat ion were reported to be unstable. I t was shown tha t AMP is the direct source of IMP, and tha t ATP can be recovered unchanged from the reaction mixture 3. The present report describes properties of the purified enzyme.

Adenylate deaminase from calf-brain homogenate was purified by high-speed centrifugation, l i thium sulfate fractionation, heat t reatment , and density gradient centrifugation. The highest purification obtained was 79-fold, with a recovery of 640/0 . The enzyme is soluble at each stage of purification, and is stable on prolonged storage at ~L °. Solutions of adenosine t r iphosphate-act ivated adenylate deaminase are subject to sudden loss in act ivi ty unless sulfhydryl compounds such as mercapto- ethanol or glutathione are present.

The purified enzyme does not show an absolute dependence on ATP. The act ivat ing effect of AT P diminishes with increasing concentrat ions of AMP. A plot of AMP concentrat ion against enzyme act ivi ty in the absence of ATP shows a sigmoidal relationship, whereas in the presence of ATP it shows a hyperbolic relationship (Fig. I). Vmax in the presence and absence of ATP is the same. When the results obtained in the absence of AT P are plot ted in terms of I/V against I/[SJ, a curve results, but when they are plot ted in terms of I/V against I/[Sj 2 a straight line is

3.5

3.0

2.5

E 2.0

-~ 1.5 E

-o 1.0

E 0.5

~ , Z

q

i i i i

/ lb ~o 3'o 4b

[AMP] (raM) 50

Fig. I. Effect of AMP concentration on adenylate deaminase from calf brain ill the absence (O--O) and presence (O---O) of 5 mM ATP. The reaction mixture also contained 15 mM reduced glutathione, 37-5 mM Tris-HC1 buffer (pH 7.2), and enzyme. The temperature of incubation was 37 °, and samples were analyzed for ammonia 13 produced 3,8, and 13 min after starting the reaction.

* Publication No. 336 of the Graduate Department of Biochemistry, Brandeis University•

Biochim. Biophys. Acta, 96 (1965) 535-537

536 PRELIMINARY NOTES

obtained. When the results obtained in the presence of ATP are plotted in terms of I/V against I/[S] a straight line results (Fig. 2). In the absence of ATP, one-half Vmax occurs at lO.4 mM AMP, whereas in the presence of 5 mM ATP, one-half Vmax occurs at 1. 7 mM AMP. Thus at one-half Vmak, ATP increases the apparent affinity of the enzyme for AMP about 6-fold. The maximum stimulation by ATP (activated rate/non-activated rate) occurs at AMP concentrations less than 2 mM (Fig. I). When the results shown in Fig. i are plotted in terms of log (v/Vmax -- v) against log[S] (ref. 4) two straight lines result. The slopes of these lines are 2.1 and 1.2 in the absence and presence of ATP, respectively.

Solutions of the enzyme are inactivated by heating at 5 °0 for 30 min. Such heat inactivation can be largely prevented by 15 mM ATP. Tripolyphosphate and AMP also protect against heat inactivation of the enzyme, but they are less effective than ATP.

lAMP] -1 (raM) -1 (c--~)

or" [AMP] -2 (raM) -2 in obsence of ATP(o---<)) =o

2 . ; , . i i i i

"7

2.0

1.5

x~ 1.0 o E t_ ~o £ 0.5 Z v !

- Q 5 0 EAMP] -1 (raM) -1 in pPesence of ATP t:

0.05 0.1 0.1.5 0.2 0.25

/: /

d5 1'.o ~'5 2'o 2.5 :)

Fig. 2. P lo t of I/V aga ins t I/[S] (solid lines) a n d I/[S] 2 (broken line). The d a t a f rom Fig. I have been replot ted . O - - O a n d O . . . . O, A T P absen t ; O - - O , A T P present .

The experimental results can be most simply explained by assuming that the enzyme occurs in an active and an inactive form, and that it has at least two sites for AMP. Occupation of one AMP site of the active form of the enzyme by substrate prevents reversion of the enzyme to the inactive form. This is equivalent to increasing the number of active sites, or to an increase in the apparent affinity constant for AMP. Under certain circumstances this type of behavior will yield a straight line when plotting I/V against I/[S] 2, and it may yield a value of n = 2.0 when plotting l o g ( v / V m a x - v) against log[S]. The enzyme must also have at least one site for ATP. I f both of the AMP sites are "active", then the ATP molecule must occupy a third and separate site, because Vmax of the reaction is identical in the presence and absence of ATP. I f only one of the two AMP sites is "active", then the second site for AMP and the site for ATP may be identical. The evidence presented does

Biochim. Biophys. Acta, 96 (I965) 535-537

PRELIMINARY NOTES 537

not permit a distinction between these possibilities, but a separate site for ATP is the more likely in view of the lack of activation of the enzyme by ADP 3. Protection by ATP against heat denaturation suggests that the active form of the enzyme is the more stable.

The properties of adenylate deaminase from calf brain are in many ways similar to those of aspartate transcarbamylase 5, threonine deaminase 4, and the DPN-spec:ific isocitrate dehydrogenases e-l°. Hemoglobin is the best known example of an enzyme in which combination of substrate with one binding site increases the the apparent affinity for substrate of other binding sites in the same moleculeS, n. The crystal structure of oxyhemoglobin has actually been shown to be different from that of hemoglobin 1.. A discussion of these and other examples has been presented by MONOE,, CHANGEUX AND JACOB 4,13.

This work was supported by the U.S. National Institutes of Health (GM- 07261-04).

Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. (U.S.A.)

BARBARA CUNNINGHAM

J.~ M. LOWENSTEIN

I J. A. MUNTZ, J. Biol. Chem., 2Ol (1953) 221. 2 H. WEIL-MALHERBE AND R. H. GREEN, Biochem. J., 61 (1955) 218. 3 J. MENI~IClNO AND J. A. MUNTZ, J. Biol. Chem., 233 (1958) 178. 4 J. MONOD, J . -P . CHANGEUX AND F. JACOB, J. Mol. Biol., 6 (1963) 306. 5 J. c . GERHART AND A. B. PARDEE, Federation Proc., 23 (1964) 727 • 6 J. A. H&THAWAY AND D. E. ATKINSON, J. Biol. Chem., 238 (1963) 2875. 7 B. D. SANWAL, M. W. ZINK AND C. S. STACHOW, J. Biol. Chem., 239 (1964) 1597. 8 R. F. CE[EN AND G. W. E. PLAUT, Biochemistry, 2 (1963) lO23. 9 R. F. C~mN, D. M. BROWN AND O. W. E. PLAUT, Biochemistry, 3 (I964) 552.

io H. GOEBELL AND M. KLINGENBERG, Biochem. Z., 34 ° (1964) 441. 1I Q. H. GIBSON, Progr. Biophys. Biophys. Chem., 9 (1959) i . 12 M. F. PERUTZ, Proteins and Nucleic Acids, Elsevier, A m s t e r d a m , 1962, p. 37. 13 J . -P . CHANGEUX, Broohhaven Symp. Biol., 17 (1964) 232. 14 P. B. n2twK, n . L . OSER AND W. H. SUMMERSON, Practical Physiological Chemistry, The

Blak i s ton Co., Phi lade lphia , 1949, p. 818.

Received .January 8th, 1965

Biochim. Biophys. Acta, 96 (1965) 535-537

PN 61049 Conformation changes of yeast phosphopyruvate hydratase (enolase)in- duced by activating and inhibiting metal ions

The usefulness of difference spectroscopy for measuring the binding of activating metal ions 1 and substrate 2 to enzymes has recently been demonstrated: We are reporting changes in the spectrum of yeast phosphopyruvate hydratase (2-phospho- D-glycerate hydro-lyase, formerly known as enolase, EC 4.2.1.11) upon binding of Mg 2+, Mn '~+, Zn z+, Cd ~+, and Hg ~+ that indicate conformational changes. Under the conditions of this study Mg 2+ only activates the enzyme, while Mn ~+, Zn 2+, and Cd ~+

Biochim. Biophys. Acta, 96 (1965) 537-54 o