binding and structural proteins [Berg, 1986; Coleman, 1992; Klug and Rhodes, 1987]. Confirmation of the involvement of this region in zinc chelation has recently been obtained
by m utagenesis studies w ith the m agnesium dependent ALAD from B. ja p o n icu m
[Chauhan and O'Brian, 1996] and this is discussed in section 1.3.2.2.
The nature of the two zinc binding sites in bovine ALAD has been investigated by extended X-ray absorption fine structure (EXAFS). This revealed that the non catalytic zinc (term ed Zne or |3) has a tetrahedral co-ordination of four cysteines w hereas the catalytic zinc (termed Zn^ or a) is bound to two / three histidines, one/zero oxygen from a
group such as tyrosine or water, one tyrosine or aspartate and one cysteine [Dent et aL,
1990]. Sequence localisation of the im portant cysteines has been attem pted and is
sefluSACo.
discussed in section 1.3.4. In addition, a pileup show ing the possible sequence localisation of all the residues involved in the chelation of each type of zinc is shown in figure 4.8. The putative roles of the zinc ions will be considered in relation to the enzyme active site and mechanism in section 1.3.3.
E. coli ALAD has a zinc binding motif and is zinc dependent [Spencer and Jordan, 1993; Mitchell and Jaffe, 1993] but has some unusual characteristics; The enzyme binds catalytic and non catalytic zinc at a stoichiometry of two zinc per subunit i.e. sixteen per octamer [Spencer and Jordan, 1993] which is tw ice the num ber bound by the m am m alian enzym es. In addition, the activity of E. coli ALAD with zinc bound is stim ulated by
m agnesium [Spencer and Jordan, 1993; M itchell and Jaffe, 1993]. E. coli A LAD may
hence be better described by a class separate to the solely zinc dependent ALA Ds but it would be im portant to re-exam ine metal binding in mammalian ALADs before making such a division.
The discovery of the magnesium stimulation of the zinc dependent E. coli ALAD led Jaffe (1993) to propose a model describing three metal binding sites in ALADs called A, B and C. In this work, the sites are referred to as a, p and C in order to avoid confusion with the
A and P side ALA as suggested by Spencer and Jordan, [1994]. It is proposed that
mammalian ALADs, as discussed above, bind zinc at sites a and p and contain positively
charged amino acids in place of site C, whereas E. coli binds zinc at sites a and p and
m agnesium at site C [Jaffe, 1993]. The model in its most recent form is shown in table
1.2 [from Petrovich et al., 1996] and it incorporates classification also of the magnesium
dependent ALADs w hich will be discussed in section 1.3.2.2. The model has not been accepted by all investigators and discussion centres around whether magnesium binds to E. coli ALAD solely as an allosteric activator in the site designated C or w hether it can substitute directly for som e of the zinc bound at sites a and p. In particular, the stoichiometry o f zinc binding to E. coli ALAD proposed by Jaffe and shown in table 1.2
Chapter 1 : Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
is not in accordance with the sixteen moles of zinc per octamer of enzyme determined by Spencer and Jordan, 1993. Metal binding stoichiometries and possible modifications to Jaffe's model for ALADs will be discussed further in chapter 4 in the light o f results obtained during this work which reveal that E. coli ALAD is not alone in its ability to bind both magnesium and zinc, this is a characteristic shared at least with the ALAD from yeast.
T a b le 1.2. T h e p ro p o se d th re e m e ta l b in d in g site m odel fo r A LA D s [fro m P e tro v ic h et aL, 1996].
A LA D Site A f a ) .
(Metal and no. o f ions required per octamer for full activity)
Site B fp ).
(Metal and no. o f ions required per octamer for full activity)
Site C.
(Metal and no. o f ions required per octamer for full activity)
Mammalian 4Zn(H ) 4 Zn(n) Absent
E. coli 4Zn(II) 4 Zn(II) 8 Mg(II)
Plant 4M g(II) 4 M g(n) 8Mg(II)
B. japonicum 4 M g (n ) Absent 8Mg(II)
In addition to metal binding variances, the yeast and E. coli ALADs also have more alkaline pH optima than their animal counterparts, 9.8 for the yeast enzyme and 8.5 for the enzyme from E. coli [Borralho et a l, 1990; Spencer and Jordan, 1993]. However, differences in 'pH optima' between the enzymes may be misleading since investigators have measured a 'rate' of reaction at different pH which is a combination of the changes in enzyme and Vmax (see section 4.2.10).
1.3.2.2) M a g n e siu m d e p e n d e n t A LA D s.
The less studied magnesium dependent class of ALADs includes the plant ALADs from spinach [Schneider, 1970], Nicotiana tabacum [Shetty and Miller, 1969], wheat leaves [Nandi and W aygood, 1967] and pea [M.P. Timko, pers. commun.] as well as the bacterial ALADs from M ycobacterium phlei [Yamasaki and M oriyama, 1971] and
Bradyrhizobium japonicum. [Petrovich et a l, 1996]. In addition, sequence conservation
has led to the inference of magnesium dependency for the ALAD from Chlamydomonas
reinhardtii [Matters and Beale, 1995].
Studies on the ALADs from N icotiana tabacum and wheat showed that activity of magnesium dependent ALADs can also be supported by manganese [Shetty and Miller,
Chapter 1 : Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
1969; N andi and W aygood, 1967]. M anganese activates the enzym es at low concentrations but becomes inhibitory at around 2.5 mM. The importance o f this binding is unclear although a typical plant cell contains 0.06-0.6 mM Mn and 15-60 mM M g in the cell sap and 0.01 mM Mn and 1.5 mM Mg in nutrient solution, suggesting that either of these metals could have a physiological role [Hewitt and Smith, 1975]. The effect of Mn on ALADs is investigated in section 4.2.7.
The m agnesium dependent enzymes are reported to have alkaline pH optim a of approximately 8.0-8.5 [Yamasaki and Moriyama, 1971; M.P. Timko, pers. commun.], but these values were determined by measurement o f an average rate o f reaction as described above.
Spinach ALAD is apparently a homo-hexamer [Schneider, 1970] whereas the ALAD from
B. japonicum is octameric [Petrovich et aL, 1996] and work presented in this thesis
shows that the ALAD from pea is also octameric. The multim eric nature o f other magnesium dependent ALADs have not been reported.
The metal binding stoichiometries of magnesium dependent ALADs are also ill defined. Pea ALAD has been suggested to bind eight magnesium ions i.e. one per subunit [M.P. Timko, pers. commun.]. In addition, the ALAD from B. japonicum has been reported to bind a total of twelve magnesium ions per octamer of enzyme [Petrovich et a i, 1996]. As shown in table 1.2, these results have been interpreted as supporting the presence of four catalytic magnesium ions per octamer (at site a) and eight allosteric magnesium ions per octamer (at site C). It is also proposed that site p (identified as containing the non-catalytic zinc in bovine ALAD in section 1.3.2.1) is absent and that the enzyme is additionally stimulated by potassium ions.
Stimulation of enzyme activity by potassium and other monovalent cations has also been reported for the ALAD from R. sphaeroides [Nandi et aL, 1968a]. This enzyme has been reported to be allosterically activated by K+, Li+, Rb+ and NH4+, showing a hyperbolic saturation curve with respect to these ions. The addition of K+ to the enzyme caused formation o f an equilibrium mixture of monomers, dimers and trimers, all of which were catalytically active. A similar observation was made for the ALAD from B. japonicum
although the smaller molecules were reported to be a mixture of catalytically active and inactive species [Petrovich et aL, 1996]. Unlike the ALAD from B. japonicum, however, magnesium was not reported to activate the ALAD from R. sphaeroides [Nandi et aL,
1968a]. However, the cloning of the gene encoding R. sphaeroides ALAD has been reported [Delaunay et aL, 1991] and sequence analysis reveals that the enzyme contains the m agnesium binding consensus region identified in other m agnesium dependent
Chapter 1: Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
ALADs (discussed below), [J. Zeilstra-Ryalls, pers. commun.]. It is possible that the magnesium dependency of this enzyme was initially overlooked and the ALADs from R.
sphaeroides and B. japonicum may be very similar.
At the present time it is difficult to conclude whether the magnesium binding stoichiometry and potassium stimulation of the ALAD from B. japonicum warrant its classification into a new class o f ALADs, possibly shared with the ALAD from R. sphaeroides, but separate from other m agnesium dependent ALADs. Potassium stim ulation may have been overlooked for m agnesium dependent and also zinc dependent A LA D s as the concentration of potassium in the buffers commonly used in enzyme assays exceeds 0.1
M which was reported to cause the maximum effect for B. japonicum ALAD [Petrovich et al., 1996]. Careful re-examination of ALADs from different sources will be required in order to determine whether potassium stimulation is the exception or the rule for ALADs.
Sequence analysis of the magnesium dependent ALADs reveals an overall 46% amino acid identity between, for example, pea ALAD and the highly conserved mammalian ALADs, but the zinc binding motif is missing. As shown in figure 1.9 and later in figure 4.8, amino acids 186-206 in the pea ALAD which correspond to the zinc binding motif in mammalian ALADs show substitutions o f cysteines and histidines to threonine at 187, alanine at 190 and aspartate at 192, 197 and 200 [Boese et al, 1991]. Threonine and alanine are neutral residues but aspartate is negatively charged at cellular pH and this altered arrangement of charges may aid the chelation of magnesium. W hilst the metal binding domain of these enzymes does not resemble the so-called zinc finger, it is similar to the 'EF' hand consensus sequence found in calcium and magnesium binding proteins such as calmodulin, troponin C and phospholipase D [Moncrief et a i, 1990].
The localisation o f residues involved in m agnesium binding to the sequence area discussed above has recently been confirmed by an elegant experiment involving the mutagenesis o f B. japonicum ALAD. Three proximal amino acids conserved in plant ALADs were changed to cysteine residues as found in the zinc dependent mammalian ALADs [Chauhan and O'Brian, 1996]. The residues changed were alanine 146, aspartate 148 and aspartate 156 and additionally, phenylalanine 150 was changed to tyrosine which is present in mammalian ALADs at the equivalent position. The result was a variant of B.
ja p o n ic u m ALAD which was catalytically com petent and able to support normal
nodulation but was zinc dependent instead of magnesium dependent. This suggests that one or more of the changed residues must be involved in magnesium chelation and also that some of the cysteines introduced are likely to be involved in zinc chelation as already suggested for mammalian ALADs.
Chapter 1 : Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
The other area of sequence divergence of plant ALADs from their mammalian counterparts is at the N (amino) terminus. Both the pea and spinach ALADs appear to have chloroplast transit peptides in this region although a full length clone has not been obtained for pea ALAD [Boese et aL, 1991; Schaumberg et aL, 1992]. These sequences show a net positive charge, absence o f tryptophan and tyrosine, occurrence of proline and a relative abundance of hydroxylated amino acids and this is typical of transit peptides [Boese et aL,
1991; Schaumberg et aL, 1992; Phua et aL, 1989]. In addition, the hydrophobic nature of this region suggests that these plastid localised enzymes may be at least transiently embedded in the chloroplast thylakoid membrane [Nasri etal., 1988].
The final difference between the ALADs is that plant ALADs are reportedly less sensitive to oxidation and sulphydryl modification than their animal counterparts [Liedgens et aL,
1983]. This is presumably due to the fact that cysteines are not involved in metal chelation in these enzymes.
T a b le 1.3. S u m m a ry o f th e p r o p e r tie s o f th e tw o p r in c ip a l p ro p o s e d c lasses o f A L A D s.
Z n d e p e n d e n t A LA D s M g d e p e n d e n t A LA D s
Source Mammals; avians;
some cyanobacteria; some bacteria Plants; some cyanobacteria some bacteria Metal binding motif
'Zn finger' of cysteines and histidines.
'EF' hand o f alanines and aspartates.
Quarternary Structure
Homo-octameric Homo-hexameric or
homo-octameric pH dependence Optimal rates of catalysis
at pH 6.3-7.1
Optimal rates of catalysis at pH 8.0-8.5
Sulphydryl sensitivity
Very sensitive to oxidation or modification
Less sensitive
Other properties Inhibited by lead
Mg binding also in some ALADs.
Activated by Mn. N terminal chloroplast transit peptide.
1.3.2.3) M e ta l in d e p e n d e n t A LA D s.
There may be a class of ALADs which do not require metal ions for activity. ALADs from the aerobic photosynthetic bacterium Erythrobacter sp. strain O C hl 14 [Shioi and Doi, 1988], the facultative photosynthetic bacterium Rhodobacter capsulatus [Nandi and
Chapter 1 : Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
Shemin, 1973] and the anaerobic photosynthetic bacterium Chlorobium vibrioforme [Rhie
et a l, 1996] are reported to be active in the absence of metal ions. Whilst it is possible that
there is such a class, consideration of the proposed reaction m echanism discussed in section 1.3.3 makes it more likely that metal dependency in these enzymes was not detected due to high affinity binding or that this is a very minor class restricted to a few photosynthetic bacteria.
1.3.3) T h e re a c tio n m e ch a n ism o f A LA D .
ALAD catalyses the Knorr type condensation o f two molecules of ALA to form the m onopyrrole PBG in an essentially irreversible reaction involving the form ation or cleavage of at least eight chemical bonds. The mechanism of reaction is believed to be fundamentally conserved between all ALADs, regardless of metal binding class, but a number o f different mechanisms have been proposed and it is difficult to differentiate between these in the light of limited conclusive experimental evidence. M ost o f the evidence collected concerns mammalian ALADs in which each enzyme active site binds two ALA and two Zn(II), with the zincs in one of two distinct environments [Dent et a l,
1990].
ALADs are unusual in that they catalyse the condensation between two molecules o f the same substrate (ALA), and hence the order of substrate binding cannot be determined by classical steady state kinetics. As it is known that the identical ALA molecules are distinct when bound to the enzyme and their chemical fates are different, PBG having two sides which originate from different molecules of ALA: the acetic acid or 'A' side and the propionic acid or 'P' side as shown in figure 1.10, a knowledge of the order o f substrate binding is crucial for the elucidation of the enzyme mechanism. The order for ALADs has been investigated [Jordan and Seehra, 1980; Jordan and Gibbs, 1985] by single turnover experiments using isotopically labelled substrate.
The single turnover experiments demonstrated that the ALA which can bind initially to the enzyme gives rise to the side o f the product. This is hence referred to as the P -side ALA and it forms a Schiff base to Lys 252 of mammalian ALADs [Nandi and Shemin, 1968c; Gibbs and Jordan, 1986] or the equivalent Lys 319 in pea ALAD [Boese et aL,
1991]. The origins o f the P and A sides of PBG are illustrated in figure 1.10 and proposed reaction mechanisms are shown in figure 1.11.
Chapter 1: Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase