SECTOR NORTE PAYLLA
4.2.4. Estructura del ritual: Las deidades
Samples of the first deaminase-containing fraction eluted from the f.p.l.c. of D84A, D84E and D84N were subjected to electrophoresis on a non denaturing polyacrylam ide gel. Therefore, in the case of D84A and D84N, it was not the fractions which contained the majority of the enzyme that were taken bu t those that gave rise to an earlier smaller deaminase peak (i.e., the fractions which w ere proposed to contain free enzyme). For comparison, the fractions were ru n alongside samples of apoenzyme and wild-type
deaminase.
The same sam ples were also incubated w ith a thirty m olar excess of porphobilinogen at 37°C for 10 minutes. These mixtures were subject to electrophoresis on a non-denaturing polyacrylamide gel. The results are show n in figure 3.20.
The non-denaturing gel in figure 3.20 shows apoenzyme appearing as a ladder of bands that m igrated behind the wild-type, whilst w ild-type
m igrated w ith the typical double band profile. The D84A and D84N m utants appeared to have the same mobility, and m igrated to the same distance as the higher band (less mobile) of wild-type. The D84E m utant appeared to migrate slightly beyond the D84A and D84N m utants and to a distance in betw een the double band of wild-type. The mobility of the m utants is consistent w ith their respective charges, is identical to the mobility previously observed for the m utants purified under reducing conditions and are the results expected for the holoenzyme form of the m utants.
This suggests that the two different mobilities previously observed for the D84A and D84N m utants purified in the presence and absence of reducing agent (Woodcock, 1992), w hich m igrated just behind D84E and m uch further than D84E respectively, in fact indicated two forms of the enzyme that do not appear independantly of one another but are both present in the cell. In other words, it indicates that the enzyme exists as the holoenzyme form and an
Chapter 3 : Characterisation of the Asp-84 Mutants
e n z y m e -in te rm e d ia te co m p lex form . T h e co m p lex , in w h ic h fo rm th e m a jo rity of th e D 84A a n d D 84N e n z y m e ex ists, is p re d ic te d to b e th e ES2 c o m p le x a n d th e p re v io u s o b se rv a tio n s of it a p p e a rin g o n ly in th e ab sen ce o f re d u c in g a g e n ts are m o re likely to b e d u e to th e p u rific a tio n te c h n iq u e r a th e r th a n th e o x id a tio n sta te of th e cofactor.
F ig u re 3.20 T he N o n -D e n a tu rin g P o ly a c ry la m id e G el C o m p a rin g th e D 84A. D84E a n d D 84N M u ta n ts of P o rp h o b ilin o g e n D ea m in a se
L ane 1 c o n ta in s a p o e n z y m e (w ild -ty p e con trol).
L an e 2 c o n ta in s w ild -ty p e h o lo e n z y m e (w ild -ty p e contro l).
F o r c la rity , th e a p o e n z y m e , h o lo e n z y m e , a n d th e b a n d s w h ic h m ig ra te b e y o n d th e h o lo e n z y m e a re m a rk e d in th e fig u re .
L an e 3 c o n ta in s m u ta n t D84A. L ane 4 c o n ta in s m u ta n t D84E. L an e 5 c o n ta in s m u ta n t D 84N.
L an e 6 c o n ta in s w ild -ty p e a p o e n z y m e in c u b a te d w ith p o rp h o b ilin o g e n . L ane 7 c o n ta in s w ild -ty p e h o lo e n z y m e in c u b a te d w ith p o rp h o b ilin o g e n . L an e 8 c o n ta in s m u ta n t D 84A in c u b a te d w ith p o rp h o b ilin o g e n .
L an e 9 c o n ta in s m u ta n t D84E in c u b a te d w ith p o rp h o b ilin o g e n . L an e 10 c o n ta in s m u ta n t D 84N in c u b a te d w ith p o rp h o b ilin o g e n . 1 2 3 4 5 6 7 8 9 10
I
- a p o e n z y m e < --- h o lo e n z y m e (E) — b a n d s m ig ra tin g b e y o n d h o lo e n z y m eT h e g el in fig u re 3.20 also gives d e ta ils of th e effect of s u b s tra te in c u b a tio n w ith a p o e n z y m e , w ild -ty p e h o lo e n z y m e a n d th e a sp a rta te -8 4
m utants. The apoenzyme still appears to electrophorese as a ladder of bands as previously observed (Scott et ah, 1989; H art et al., 1988), and now also contains a num ber of bands that run beyond the w ild-type double band. The w ild-type enzym e also displays a num ber of extra bands that run beyond the double band. The D84A and D84N m utants do not display any extra bands. The D84E m utant displays only one strong band which migrates to the same distance as the lowest new band observed for wild-type.
The w ild-type apoenzyme therefore appears to have reconstituted into holoenzyme after incubation at 37°C w ith porphobilinogen, and has
accumulated as enzyme-intermediate complexes. The wild-type has also m ade enzym e-interm ediate complexes on incubation w ith porphobilinogen.
The D84E m utant is found to have turned completely to an enzyme- interm ediate complex after 10 minutes incubation at 37°C w ith
porphobilinogen. The D84A and D84N m utants are found to be inactive on incubation w ith porphobilinogen, in agreement w ith the fact that they are catalytically inactive. Thus the intriguing question rem ains as to how these m utants have formed as ES2 complexes.
3.2.5.2 Spectral Properties of the D84A, D84E and D 84N M utants