Abstract
PEPCase is a regulatory enzyme of C4 photosynthesis. The encoding region 111 the gene has a conserved 3' region containing the activie site, and a variable 5' regulatory region. Deletions to the 5' region could potentially produce functional, but deleted clones that can be used for investigating the role and function this region in protein catalysis and regulation.Two full length PEPCase cDNA clones from Flaveria trinervia (C4), Clones 5 and 7, were selected for this study. Clone 7 exhibits 5 times the PEPCase activity of Clone 5, which has an extra 70-100 bases on the 3' end of the cDNA insert. Digestion of these full length clones with the restriction enzymes Bsi EI, Bsm AI and Nhe I produced new PEPCase inserts that were 79, 477 and 974 bases shorter at the 5' ends than their "parent" full length clones (Clones 5 and 7) respectively. The first two deleted inserts were blunt-end ligated to the expression vector, Vector 4.4. The third deleted PEPCase insert (Nhe I), was ligated to the expression vector, Vector 2.5, which was also derived from the pSI 4001 vector.
Only three out of the six possible clones were isolated. These are the Deleted Clone 5 Nhe I (974 bases shorter), Deleted Clone 7 Nhe I (974 bases shorter) and Deleted Clone 7 Bsi EI (79 bases shorter). The enzymic properties of these truncated PEPCase proteins were investigated and comparisons made with the full length clones from Clones 5 and 7 from which they were derived. The effect of pH, malate, G6P and PEP concentrations were examined.
In general, the activity of the deleted clones was approximately 1/5 of the activity of the full length clones, indicating that deletions had greatly diminished catalytic activity. This could be due to a number of reasons. Firstly the deletion of 974 bases of Nhe I would remove many basic amino acid residues thought to be involved in the binding of PEP (Andreo et al., 1987). This would result in the altering of the active site and a decrease in activity. Secondly, theNhe I deletion removed residues involved in the maintenance of the tetrameric structure of PEPCase (Walker et al., 1986). Lastly, the decrease in PEPCase activity could conceivably be partly due to the dissociation of the enzyme into dimers or monomers.
The pH optimum for all the deleted clones was pH 7.6, which was similar to that of the full length clones. The enzyme from clone 7, full length or deleted, appeared to be more responsive to pH change than those from clone 5. Since the same vector was used for Deleted Clone 5 Nhe land Deleted Clone 7 Nhe I, the differences in PEPCase activity may possibly, reflect intrinsic differences between clone 5 and 7 in the remaining coding regions.
At pH 7.6, the affinity for PEP obtained for PEPCase from the Full Length Clone 5 was higher than that of the Deleted Clone 5 Nhe L In contrast, the deleted clones of Clone 7 exhibited a higher affinity for PEP than the Full Length Clone 7. Thus the same deletion produced completely different results in Clones 5 and 7. This difference may again be associated with the intrinsic differences between Clones 5 and 7.
Malate strongly inhibited the full length clones and abolished activity in the Deleted Clone 7 Bsi EI In contrast, it appeared to have a negligble effect on both Deleted Clones 5 and 7 Nhe I. The Bsi EI deletion would remove the serine residue responsible for protein phosphorylation from the 5' region. This serine is thought to confer protection for the enzyme against malate inhibiton. Its deletion by Bsi El would presumably remove protection against malate and greatly enhance malate inhibition. Deletion by Nhe I would remove 974 bases from the 5' regulatory region which contained the serine residue. The apparent lack of malate inhibition on this deleted clone suggests that this deletion might have also removed the malate binding site. This would suggest that the malate binding site lies in the region between the Nhe I and Bsi EI restiction sites.
G6P increased PEPcase activity and increased affinity for PEP in all clones examined. However, the extent of activation varied between the PEPCase clones. Activation of G6P was reduced in Deleted Clone 5 Nhe I, compared to the Full length Clone 5. These results partly support the hypothesis by Wedding et al., (1989) that G6P binds to cysteine and histidine residues in the regulatory region. Thus deletion of these in the Deleted Clone 5 Nhe I would cause a decrease in G6P activation. In contrast, the increase observed in the Deleted Clone 7 Bsi EI and Nhe I clones were greater than that obtained in the Full Length Clone 7. These results are different to those observed for the Clone 5 and, again, imply that there are intrinsic differences between Clones 5 and 7. These differences were presumably masked by the 5' regulatory region in the full length clones, but only came apparent when this region was removed by Nhe I or Bsi EI deletions. When put together these results suggest that G6P binds to the 5' regulatory region as suggested by Wedding et al. ( 1889), and also to the conserved region.
This study indicates the value of using deleted clones to evaluate the relationship between structure and function in PEPCase and to elucidate possible regulatory mechanisms asociated with the regulation of PEPCase. Results have shown that deletion of up to a third of the PEPCase enzyme did not abolish catalytic activity or the effects of modulators such as G6P and malate. The results indicate the importance of the 5' regulatory region in the regulation and catalysis of PEPCase and emphasise that intrinsic differences between PEPcase clones may also play an important role in the regulation of PEPCase. Finally, the results suggest that malate and G6P bind at different sites on the PEPCase enzyme and that the malate binding site may possibly lie between the Bsi Eland Nhe I restriction sites on the 5' region, while G6P appears to bind at two sites - the 5' regulatory region and the 3' conserved region.
| Date of Award | 1993 |
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| Original language | English |