A.L. GUARDIOIA, D. PARETSKY AND LV. l l c E \ w ~
418
[COSTRIBUTION FROM
THE
DEPARTMENTS O F BACTERIOLOGY AND
CFIE?JISTRY.
CXIVERSITY
Vol. 50 OF
KANSAS]
The Interaction of DPN+' with p-Aminobenzoic Acid and Analogous Compounds
After D P N + was caused t o react with either P-lBAk,lor methyl p-aminobenzoate, p-diinethylaminobeuzoic acid, methyl p-dimethylaminobenzoate, m-aminobenzoic acid, o-aminobenzoic acid and folic acid, it was no longer susceptible to cleavage by DPNase. When the amino group of PAUL?, was acetylated, the resistance to cleavage was greatly reduced. 3,5-Diinethyl-~-aiiiinobenzoicacid produced no inhibition of cleavage. When (DPN.CX\T)'was used, no inhibition was observed. It is suggested that a chemical reaction takes place between D P N + and PBB.2 forming a new compound upon which DPSase is no longer active. -4 inechanism for the re:iction is postulated, based 011 the resonance o f the pyridiniuni inviety of the IIPN?,
Introduction 111 an attempt to extend the work of LVoolley?
et ~ l . , ' - ~on the role of "onium" compounds in metabolism, PABA was selected originally as a possible acceptor amine for the DPN+-DPNase system. It was found that instead PABA inhibited the cleavage of D P W by DPNase. I n this report data are presented 011 the nature of the ap9 1
'
lNHIElTllJN
OF T H E C L E A V A G E O F D P N ' /
i / / /
/ I1
IPABArDPNarcI,-DPN'
21
(PAL)A+DPNI
7
3) ( D P N ' t OPNoae
> 2
c-
- 340
DPNaic 4
PABA1
mp
__
,
10
I
"m
Experimental and Results During the course of studies on the role of "onium" compounds in metabolism, 20 p m . of P-kB.1 was treated with 9 pm. of D P X + in the presence of DPNase. It was observed that P.4BX apparently inhibited the DPNase activity, as measured by cleavage of I)PN+. In an attempt to determine the nature and mechanism of this inhibition, varying amounts of P.1B.i ranging froin 0 to 50 p m . were incubated with (a) 1 unit DPSase,7 (b) 9 pm. D P S + and (c) 9 pm. D P N + 1 unit DPSase. I t the end of the incubation period, 9 pm. D P N + and 1 unit DPSase were added, respectively, to systems (a) and (b) and incubated for another 21 hours. The results obtained are shown in Fig. 1. The optical densities are inversely proportional to the activity of DPNase; that is, the greater the activity of DPSase, the greater the cleavage of D P N + and hence, the lower the optical density a t 340 mp. It can be seen that the maximum inhibition was obtained a t a P;1BX concentration of 20 pm.; the enzyme is not inactivated: if i t were, a linear inhibition should have been obtained in system (a). It thus seemed that PABA and DPN' may have combined in some manner, thus preventing the enzyme from cleaving the latter. In an attempt t o determine the site of reactivity of the PABA molecule, its functional groups were blocked. pDimethylaminobenzoic acid, methyl p-arninobenzoate aiid methyl p-dimethylaminobenzoate were tested for inhibitory action. The results obtained showed that each one of the compounds tested caused effective inhibition of enzyme action with an optimum inhibition at a concentration of 20 p m . of analog. These observations made it clear that neither the carboxyl nor the amino groups were directly involved in the reaction. In addition to these data, microbiological assays of the reacted mixtures showed that PAB.1, as such, did indeed disappear, yet the Bratton-Marshall testQfor amino groupings indicated an apparent presence of P-1B-1. Resonance theory was used in an attempt t o reconcile the apparent contradictions in the data. The amino groiip of all the compounds investigated possesses an unshared pair of electrons which can enter into resonance interaction wit11 a benzene ring giving rise to structures in which the orfiio and para carbon atoms bear a formal negative cliarge al!d til? nitrogen a formal positive charge. Due t o resonatlrc the positive charge of the nicotinamitle portion of the D P S ' molecule can be shared b y the 01- a n d y-carbon atoms of the ring as n-ell as by the tiitrogeti atom. \\-it11 these cotisicicrations in mind, the mechanism sliowii in Fig. 2 is proposcd. This mechanism fits all the data nrhicli have beeti obtained in previous experiments: the amino group is free to give the positive Bratton--Marshall test, yet the free PdBA has disappearcd. X known anallogy of this proposed reaction is the condensation of benzoyl chloride, dimethylaniline a ~ l d
+
/
/
The incubation of D P S +, 1)PNase a i d PABA or relatetl compounds was carried out in a water-bath a t 37" with colitinuous shaking for 24 hours a t pH 6.0.
- ~ . . Io 20 50
PABA.
Fig. 1.-Inhibition of the cleavage of D P S + by P.1B-1: temperature, 3 7 " ; incubation time, 24 hr.; PH 6.0, phosphate-citrate buffer, F/2, 1 2 ; D P N + , 9 pm./ml. (final concn ) ; PABA, data expressed as final concn.; final volume, 4 in1
parent inhibitinn of DPNase by PXRA and related compounds.
Materials and Methods The DPNase used was prepared from beef spleen3 and purified by ammonium sulfate fractionation. DPTC'ase activity !?as determined by measuring the change in optical density of the incubated s ~ s t e mat 340 mp after cyanide addition, employing a Beckman DU spectrophotometer.6 (1) D P N +, diphosphopyridine nucleotide (oxidized) ; DPNase, diphosphopyridine nucleotidase; PABA, #-aminobenzoic acid: ( D P N C S ), diphosphopyridine nucleotide-cyanide complex. (2) D. W.Woolley, Notzruz, 171, 323 (1953). ( 8 ) (a) D. W.Woolley, THISJ O U R N A L , 73, 1898 (1951); (b) S. G. A. Alivisatos and D. W. Woolley, ibid,,77, 1065 (1955). (.i) S. G. A . Alivisatos and D. W. Woolley, J . B i d . C h e m . , 2 2 1 , 651 (1956). (a) I.. J. Zatman, N. 0. Kaplari and S. P . Colowick. ibid., 200, 197 (1953). (6) S. 1'. Colowick N. 0 . Kaplan and M. Ciotti, i b d , 191, 4-17 (!M1 ) .
( 7 ) 1 unit D P S a s e is t h e amount which will cause t h e splittin!: of D P S + a t a temperature of ::7O for 24 hours. (8) S C. Agarwala and LLT. H Peter,,nn. . t r , - I i Iiiochenr.. 27, :iOI (i9m). (9) A. C. Bratt
