E nzym e Characterisation

Figure 4.9. Effect of zinc on the activity of yeast ALAD.

a) Yeast ALAD: Zn binding (o-phe chelation) 100 n ts c 8 5 0 - >

5

f?) Yeast ALAD: Zn binding (EDTA chelation)

100 -1 7 5 - ê c 5 0 - > < CN o o p H 8 .0 - A p H 6 .0 Z n ( m M )

Yeast enzym e w as treated with the minim um concentration o f (a) o-phe or (I?) ED TA required to chelate all bound m etals (0.1 m M o-phe, 10 m M ED TA ). Z inc w as subsequently added back and the activity m onitored. A ctivity is expressed as % control w here the control represents enzym e that has not been treated with chelating agent. The control sam ples were assayed in the presence o f a full zinc com plem ent and the specific activity o f tiiis sam ple was 0.99 jiM PEG s'^ m g'^.

Chapter 4: Enzyme Characterisation

The stoichiometry of zinc binding was investigated by atomic absorption and the results, shown in table 4.3, demonstrate that yeast ALAD is associated with approximately two zinc ions per protein subunit. This stoichiometry is very similar to that observed with the enzyme from E. coli.

The effect o f magnesium on the activity is shown in figure 4.10. In the absence of exogenous and endogenous zinc, the yeast ALAD displays no enzym atic activity. If optimal zinc, (0.3 mM for enzyme chelated with o-phe, 0.001 mM if chelated with EDTA), is added to apo (metal free) yeast ALAD, addition of magnesium has no effect on activity. However, if the enzyme is assayed in the presence of suboptimal concentrations of zinc, addition of magnesium increases the activity of the enzyme such that it is restored to a near maximal level. These results demonstrate that the E. coli enzyme is not, as had been thought, unique in its ability to bind both zinc and magnesium.

The stoichiometry of magnesium binding was investigated. The results shown in table 4.3 suggest that eight moles of magnesium bind per mole of octamer, i.e. one M g(II) per subunit. This supports the theory that magnesium is able only to occupy one site (a) on the yeast ALAD subunit. As found for the E. coli enzyme, magnesium exerts its effect only at pH 8.0 and does not affect the of the enzyme (table 4.3).

Collectively, these results suggest that yeast ALAD has two metal binding sites, one of which (presumably the Znp site) must bind zinc. The second site (presumably Zna) can also be filled with zinc but can be substituted with magnesium, as found for the E. coli

enzyme. However, a major difference between the yeast and E. coli enzymes is that if yeast ALAD is assayed in the presence of magnesium and an optimal concentration of zinc, no further stimulation of enzyme activity is observed. Hence it seems unlikely that the yeast ALAD has a third, stimulatory binding site for magnesium.

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Figure 4.10. Effect of magnesium on the activity of yeast ALAD.

a) Yeast ALAD: Mg stimulation (o-phe chelation) 100 75 - P p 5 0 - > < o #--- 0.02mM Zn pH 8.0 0 ... 0.05mM Zn pH 8.0 -O 0.1 mM Zn pH 8.0 O 0.3mM Zn pH 8.0 A 0.3mM Zn pH 6.0 A no Zn Mg (mM)

b) Yeast ALAD: Mg stim ulation (EDTA chelation 25 -I 75 - 50 #--- O.OOlmM Zn pH 8.0 - A O .O O lm M Zn pH 6.0 { ] ---- 0.0I m M Zn pH 8.0 No Zn Mg (mM I

Enzyme chelated with either {a) o-phe or (b) EDTA was bound with either an optim al or suboptimal concentration of zinc (0.3 mM optim al and 0.1 mM sub-optim al for o-phe chelation, 0.01 mM optim al and 0.001 mM suboptimal for EDTA chelation) and a range of magnesium concentrations added. Activity is expressed as % control where the control represents enzym e that has not been treated with chelating agent. The control samples were assayed in the presence of a full zinc complement and the specific activity of this sample was 0.99 pM PEG s'^ m g'^.

Chapter 4: Enzyme Characterisation

An examination of the primary structure of yeast ALAD reveals that the enzyme contains the putative ligands to both Z na and Znp (figure 4.8). The yeast primary structure does not contain the proposed magnesium stimulatory site identified in E. coli ALAD (figure 4.8^) and there is no other obvious magnesium binding site. The analysis of yeast ALAD activity on binding magnesium and zinc is consistent with a model in which there are only two metal binding sites (Zna and Znp) in the enzyme and the absence of a putative Mgc site is not therefore surprising. It is, however, unclear how m agnesium w ill be accommodated at the Z na site. Earlier work with E. coli ALAD suggested that this may be achieved by the change of a cysteine to a serine at position 229 [Spencer and Jordan,

1994]. However, the yeast enzyme has a cysteine at this equivalent position (position 234 in yeast sequence in figure 4.86) and this therefore seems unlikely to be the explanation, at least for yeast ALAD.

4.2.6) M e ta l b in d in g in p e a ALAD.

The metal dependency of pea ALAD has not previously been thoroughly investigated although the enzyme is anticipated to be magnesium dependent [M. P. Timko, pers. commun.]. The enzyme was therefore investigated here.

Chelation and metal binding studies were attempted as described for the yeast and E. coli

ALADs but o-phe and EDTA were found to be ineffective. However, magnesium could successfully be removed from the enzyme rapidly in a pH independent manner by the use o f Chelex binding resin (Bio-Rad) and this resulted in inactivation o f the enzyme. Activity could be restored by the addition o f magnesium revealing one type of binding site with in the order o f 0.1 mM (figure 4.11). Atomic absorption studies to determ ine the stoichiometry of magnesium binding proved impossible, presumably since magnesium is bound only loosely to the enzyme.

Chapter 4: Enzyme Characterisation