Enzyme structural changes taking place during metal binding or removal were studied by circular dichroism (CD).
CD spectra were taken of the yeast apo-ALAD backbone and aromatic regions and also of the apo-enzyme in the presence of various molar equivalents of metals. The results shown in figure 4.13 demonstrate, rather surprisingly, that no changes were detected in either region during metal binding or removal by chelation. This rules out the possibility o f any m ajor conformational change occurring on metal binding and the role o f the zinc ions in the enzyme cannot solely be a structural one because at least secondary and tertiary enzyme stmcture are maintained in the absence of metals.
Further evidence for this lack of conformational change and evidence concerning the quartem ary structure o f ALAD following metal removal was sought from 7.5% non denaturing PAGE using yeast and E. coli ALADs. E. coli holo (metal bound) ALAD runs on non denaturing PAGE as a single band although at least six other bands o f lower intensity can sometimes be seen (figure 4.14a). Treatment of the enzyme with 10 mM EDTA results in a shift between the intensity of the bands with much more o f the enzyme as the lowest Mr species. It should be possible to isolate this species and measure its Mp in order to determine whether a breakdown of the octameric enzyme is occurring but this has not been attempted. Similar native PAGE results for E. coli ALAD have recently been reported [Jaffe et al, 1995] and these were interpreted as supporting the idea o f the dissociation of native enzyme into subunits on EDTA treatment.
In contrast, yeast ALAD runs as a single band both in the presence and absence o f EDTA (figure 4.14a) and a conformational shift for this enzyme therefore appears unlikely as was suggested by the CD data. The experiments were repeated using o-phe and in this case, no shift in the banding of the E. coli enzyme was noted on metal chelation (figure 4.14a). Similarly, the stmcture of yeast ALAD appears to be unaffected by the presence of o-phe (figure 4.14a).
Chapter 4: Enzyme Characterisation
Figure 4.12. Schem atic presentation o f the m etal binding sites in ALAD.
a) E. coli ALAD
M9 c
Zn p
Proposed magnesium allosteric stimulatory site. Potential ligands include aspartate, asparagine and glutamate.
Non catalytic zinc site. Ligands include three cysteines and possibly a serine.
Catalytic zinc site which can ---' be substituted with magnesium.
Proposed ligands include histidines, tyrosines, aspartates and a cysteine or serine.
b) Y east ALAD
Znp
Catalytic zinc site.
Proposed ligands include histidines, tyrosines, aspartates and a cysteine.
Non catalytic zinc site. Ligands are proposed to be four cysteines.
Zn a
c) P ea ALAD
A single magnesium binding site is proposed although some evidence supports a second stimulatory site also. Ligands are proposed to include aspartates.
Figure 4.13. CD spectra of yeast ALAD in different metal environm ents.
Apo- yeast ALAD was prepared by treatment with the chelating agent o-phe (0.1 mM) and this reagent was then removed by gel filtration. Spectra were taken of the apo- enzyme in the presence of an increasing molar ratio of zinc and also in the presence o f an excess of magnesium. The enzyme was in CHES buffer (20 mM) in a 0.01 cm cell.
a) F ar-U V CD sp ectra.
Yeast ALAD in the absence of zinc Yeast ALAD and zinc in the ratio 1 : 0.1 Yeast ALAD and zinc in the ratio 1 : 0.5 Yeast ALAD and zinc in the ratio 1 : 5 Yeast ALAD and zinc in the ratio 1 : 1 5
Yeast ALAD with zinc and magnesium in the ratio 1 : 15 : 50
5 . < - 3 . 180 200 220 W avelength (nm) 240 260 186
b) Near-UV CD spectra.
Near-UV CD spectra were recorded using a 2 cm cell.
Yeast ALAD in the absence of zinc Yeast ALAD and zinc in the ratio 1 : 0.1 •Yeast ALAD and zinc in the ratio 1 : 0.5 •Yeast ALAD and zinc in the ratio 1 : 5 • Yeast ALAD and zinc in the ratio 1 : 15
•Yeast ALAD with zinc and magnesium in the ratio 1 : 15 : 50
2 x 10
- 1.5 X 10 2
250 270 290
Wavelength (nm)
Chapter 4: Enzyme Characterisation
The non denaturing gels were stained for activity as described in section 2.4.7 in order to determine the catalytic activity of the protein bands observed on the gels following metal chelation. However, activity was detected in all protein bands (figure 4.146) suggesting that they are all catalytically competent. However, as activity was also detected in the samples chelated with EDTA and o-phe, it seems likely that re-activation o f the enzyme had occurred, presumably due to contaminating metals in the buffer used. It is not therefore possible to distinguish between active and inactive protein using this method. A similar problem has been reported for E. coli ALAD chelated with EDTA [Jaffe et al.,
1995].
It is difficult to draw firm conclusions without further work in this area. However, it is clear from both CD and non denaturing PAGE that chelation o f metal ions from yeast ALAD does not result in any major disruption to secondary, tertiary or quartem ary structure. It has previously been suggested that a role o f the metals in ALADs is to aid structural integrity [Nandi et al., 1968a; Jaffe et ah, 1995] but this is not the case at least for yeast ALAD. Stmctural dismption appears to occur in E. coli ALAD treated with EDTA and this has been previously documented [Jaffe et al., 1995]. However, there is no evidence for such dismption following chelation o f bound metals with o-phe and this raises the possibility that the results obtained with EDTA are partially artefactual, resulting from EDTA causing conformational changes in the protein unrelated to metal removal. It would be interesting to obtain CD spectra of E. coli ALAD treated with both o-phe and EDTA to test this hypothesis.
Chapter 4: Enzyme Characterisation