Discussion of the Purification Function of Components E and S

The purification of glutamate mutase from C tetanomorphum was undoubtedly a

tedious and time consuming task. The half-life of the activity in the phosphate buffers used, at 4 °C, was ~1 day. Therefore, aii manipulations of the protein took place fast, without unnecessary delays, until each component had been subjected to one FPLC purification step. One week was typically required to isolate 10-15 units of each component per 50 g of cell paste. While developing our protocol it was necessary to compromise the yield for the purity. Later, the optimised purification procedures had to be followed methodically to minimise any losses of activity. Undoubtedly there was room for improvement, but at this stage the purity and quantity of enzyme was sufficient for the immediate purposes.

One of the advantages of the purification described above is that both components were isolated from the same cell paste. In Barker's p u h f i c a t i o n . n s

component E was essentially treated as an impurity and removed during the drastic isoelectric precipitation step (§ 1.17 Table 1.4, p. 37). In comparison with Barker's our protocols were simpler and more reproducible. The availability of the FPLC systems offered the opportunity to isolate the homogeneous components.

Chapter 3 : Results & Discussion / Purification 87 Despite the failure of the DEAE-cellulose to separate component E from (3- methyiaspartase, Barker’s experimental details were very useful during the initial stages (Table 3.1). For example all the buffers described in the original purification publications were found to be optimised. No improvement could be devised, despite our systematic efforts.

Table 3.1 Purification of glutamate mutase - initial steps

STEP (ml) Protein (mo/ml) Activity (units) Specific Activity Yield %

1. Sonicatlon (50a cell paste) 149 15.3 94±1* 0.041 100

2. Protamine Sulphate 189 5.0 41.5 0.044 44

3. Ammonium sulphate tract. (50-80%) 43 12 ** ** **

4. Dialysis 53 10.1 18.2 0.035 28

5. Fractionation on DEAE-Cellulose 9 31.4 17.0 0.59 31

Value not reproducibie-Mean of three measurements * Value could not be obtained reliably

The low mutase activities measured during the initial steps was another puzzling finding, especially in relation with Barker's early results. Buckei and Barker in 1972 made similar observations''''2 but unfortunately they did not comment on this discrepancy. The most probable explanation, as suggested by an overview of the data available, is that various impurities acted in an inhibitory manner towards glutamate mutase. In many instances the total amount of activity increased two or even three fold after the protamine sulphate cut or the DEAE- ceiiuiose fractionation. Finally, a -70% loss of activity, over the five initial steps, was typical. It has to be emphasised, however, that yields mentioned in Table 3.1, most probably underestimate the amount of mutase activity actually present.

This became apparent after every gel exclusion chromatography coiumn (p. 82), when the activities, detected for both components, increased at least 2-foid (Table 3.2). After application on the ion exchange (5-PW DEAE) FPLC system, component E was isolated in similar specific activities as in Barker's purification.''‘'®

Chapter 3 : Results & Discussion / Purification 88 The protein was homogenous and had a specific activity of 3.8 units mg-^ (Tabie 3.2).

Table 3,2 Purification o f Component E - Final Steps

STEP Volume (ml) Protein (mg) Activity (units) Specific activity Yield* % 1. G-150 3 62.5 45.0 0.72 50 2.1st FPLC on PW-DEAE 1.9 12.4 15.8 1.27 17.5 3. 2nd FPLC on PW-DEAE 1.8 1.1 4.2 3.8 5

* Values use 90 units as the total component E activity - See table 3.1

Similar amounts of both components of glutamate mutase were obtained. The totai amount of component S activity typicaiiy isolated, was slightly higher than that of component E (15-20 units per 50 g of ceil paste). Unfortunately the small protein has a shorter half-life under the specific storage conditions. Barker's accomplishment in isolating ten times more units of component S than component E, could not be duplicated. Component S was purified to homogeneity (Fig. 3.7); the overall yields seemed to be consistent, and reproducible but much lower than these of Barker's (Tabie 3.3).

Table 3,3 Purification of component S -final steps

STEP Volume (ml) Protein (mg) Activity (units) Specific activity Yield % 1. G-150 3.0 120 39.6 0.33 44 2.1st FPLC on PW-DEAE 1.9 20.0 17.5 0.87 19 3. 2nd FPLC on PW-DEAE 1.2 2.7 9.7 3.6 10

While the final details of our purification were being established, and the pure components were to be subjected to N-terminai sequencing, Buckei ef a/.^2i

published the purification of glutamate mutase from Clostridium cochiearium, a

bacterium of the same species as 0. tetanomorphum. The German group

encountered a number of difficulties similar to ours {e.g. gradual deactivation, loss

Chapter 3 : Results & Discussion / Purification___________________________ 89 interactions column and in accordance with our results lost at least -50% of its activity during this kind of manipulation (Table 2.2, p. 37).

The high specific activities reported (6 units m g-\ Tabie 1.4, p. 37) could be

attributed either to the source of the protein (C. cochlearium), or the different FPLC

columns employed. A comparison between the purification tables (Tabie 4.1 and

2.2, p. 38), however, revealed that preparations from C tetanomorphum or C.

cochlearium do not actually contain different amounts of mutase activity. For

example, after the common exclusion chromatography step (see Tables 3.2 and 2.2, p. 37), we obtained 90 units of component E (from 100 g of ceil paste), whereas Buckei using a much faster gel (the separation lasted only of few hours instead of days), obtained 130 units. Although the claim that component E isolated

from C. cochlearium was more stable than that from G. tetanomorphum was not

rigorously supported by the available data, a more thorough comparison has to be performed, especially if component E is to be cloned and overexpressed.

Our estimations concerning the molecular weights of components E and S, were also confirmed by Buckei.^i^ Advances in the molecular biology area support the notion that the large component is produced by the ceil as a monomer with a

molecular weight of 53 K D a .^22,123 j^ e value of 128 KDa reported by Barker, was

an artefact caused by the use of native gels. The above value for component E almost certainly represented an inactive complex with a composition of E2S.

It is not clear at aii if the discovery of the monomer solved aii the problems concerning the composition of the apoenzyme. If E2 was a simple dimer, due to the presence of weak interactions between two monomers, it should not have been observed in the first place under the denaturing conditions of the SDS-PAGE electrophoresis. A similar example Is p-methylaspartase, which is known to be active as a dimer, but only the monomer can be detected on the gels (Fig. 3.4.C.3). An overview of our experiments suggested that the integrity of the E2 complex depended on both the presence of component S and the overall progress of the purification. One satisfactory explanation can be that the two monomers are connected with an S-S bridge which is somehow protected when E and S are complexed but finally breaks down after prolonged exposure to 2-mercaptoethanol. Interestingly Buckei and coworkers who used the more powerful reducing agent.

Chapter 3 : Results & Discussion / Purification___________________________ 90 1,2-dithiothreitol, throughout their purification do not mention observing any of the dimer. There was some difficulty in establishing a consistent relation between the dissociation of the dimer and loss of activity for component E. The reason for that is that the dimer is always complexed with various amounts of component S, so it is not possible to attribute the apparent increased stability of the dimer to either the presence of component S or to the existence of the dimer itself.

A few months after Buckel's report, the amino acid sequence of glutamate

mutase was published by Marsh etal. (Fig. 1.16, p. 40).^22,123 This knowledge can

be useful if significant homologies between the protein under inspection and other better studied enzymes were found, in the case of glutamate mutase a region with significant local similarities (Fig. 3.8) between component E and p-methylaspartase

was discovered and reported by Marsh and H o i i a w a y.122This observation can

quite possibly reveal the substrate (3-methylaspartic acid) binding site. Ser-173 from p - m e t h y l a s p a r t a s e , i 2 6 thought to be involved in the deamination mechanism,

is indeed included in this region. This local homology is also in agreement with the early experimental evidence that suggested the location of the glutamate binding site on component E.it^

170 190 210

p-Me Asp * * * # 0 0 0 # 0 0 0 0 0 0 * * * 0 # 0 0 'k 0 *k ic "k ^ “k "k * ^

GAEINAVPVFAQSGDDRYDNVDKMIIKEADVLPHALINWEE KLGLKG EKLLEYVK

Comp.E _{GADLLPSTIDAYTRQNRYEECE IGIKESEKAGRSLLNGFPGVNHGVKGCRKVLESVN}

90 120 140

Figure 3.8 Homologies between comp.E and Me Asp. Identical residues are

denoted by conserved by

The small subunit (14 KDa) on the other hand shows an appreciable amount (40%) of conserved positions with methionine synthase and methyimalonyl-CoA mutase from a number of sources (Fig. 3.9). Based on these data a possible role for

component S during catalysis was s u g g e s t e d .123 The small protein may be

important for the crucial binding of the coenzyme from the a-side (chapter 1). The fact that oxidation of the -SH bond did not affect the affinity for component E but

Chapter 3 : Results & Discussion / Purification 91 reduced activity,im plied an important binding role specifically for the cysteines of component S in connection with the coenzyme.

Methionine Synth. ttt QGKTNGKMVI t * ATVKGDVHDI GKNIVGWLQ tt t * CNNYEIVDLG

Hum. Methvlmal.CoA REGRRPRLLV AKMGQDGHDR GAKVIATGFA DLGFDVDIGP

Mous.Methylmal.CoA REGRRLGLLV AKMGKDGHDR GAKVIATGFA DLGFDVDIGP

P. S'il .Methylmal. CoA AEGRRPRILL AKMGQDGHDR GQKVIATAYA DLGFDVDVGP

Component S MEKKTIVL GVIGSDCHAV GNKILDHSFT NAGFNWNIG

* t t t tt** t*t* t t ttt t t* t t** t VMVPAEKILR TAKEVNADLI GLSGLITPSL DEMVNVAKEM ERQGF.TIPL LIGGATTSKA LFQTPREVAQ QAVDADVHAV GVSTLAAGHK TLVPELIKEL NSLGRPDILV MCGGVIPPQD LFQTPREVAH DAVDADVHAV GVSTHAAGHK TLVPELIKEL TALGRPDILV MCGGVIPPQD LFQTPEETAR QAVEADVHW GVSSLAGGHL TLVPALRKEL DKLGRPDILI TVGGVIPEQD VLSSQEDFIN AAIETKADLI CVSSLYGQGE IDCKGLREKC DEAGLKGIKL FVGGNIWGK

t tt *tt* t

HTAVKIEQNY SGPT...VYV QNAS.R..TV GWAALLSDT QRDDFVARTR