Comparison of PEP carboxylase from cDNA clones and leaves of Neurachne

  • Catherine Adularia Eupene

    Student thesis: Other thesis - CDU


    Phosphoenolpyruvate carboxylase (PEPC; EC is a key enzyme in C4 photosynthesis and is involved in anaplerotic reactions in C3 plants. The C4 isoform has evolved from the C form. However, the process by which this is thought to occur remains to be elucidated. A genus which contains C3, C4 and CrC4 intermediates such as the monocot genus Neurachne would provide model system for the study of the transition of C3 to C4 metabolism. 

    This study examines the biochemistry of PEPC in clones isolated from cDNA libraries of N. munroi (C4) and N. tenuifolia (C3) and in leaf tissue. Biochemical characterisation of these PEPC enzymes included response to pH, kinetic analysis and the effects of the modulators glucose-6-phosphate and malate on catalytic activity.

    The activity of PEPC from C4 leaf PEPC is 10-fold higher than that of the C3 leaf form. This higher activity corresponds to the primary role PEPC plays in carbon fixing during C4 photosynthesis. Further, the Km(PEP) for PEPC from C4 leaf is 2-fold greater than the value determined for the C3 enzyme indicating that the C4 enzyme has a lower affinity for PEP than the C3 form. The lower Km(PEP) observed for C3 enzyme would correspond to the low cellular level of PEP found in C3 leaves compared to C4 leaf tissue. The differences observed between C3 and C4 leaf extracts seem to reflect the differences in the metabolic roles of these isoforms in the tissues. 

    Fully functional PEPC cDNA clones has been isolated from N. munroi (C3.2 and CsR) and N. tenuifolia (Al, A3, A 7 and Al 0). These clones showed distinct differences in their kinetic properties indicating that there are intrinsic differences between clones as well as differences in their expression. The PEPC cDNA clones from N. tenuifolia were found to have Km<PEP) values of around 0.7 rnM while the Km(PEP) values for the C4 cDNA clones range from 0.8 to 2.8 rnM. Based on their Km(PEP) values the C4 clones can be divided into two groups : a low and a high Km group. The low Km group (Al and A3) also had higher Vmax values compared to the high Km group (A7 and AlO). 
    Glucose-6-phosphate (G6P) is an activator of PEPC. It lowered the apparent Km for PEPC from all the clones as well as the enzymes from the leaves. The observed stimulation by G6P were different for different clones. The largest increase of 156% was observed in the C3 PEPC clone, CsR, and the smallest of 66% in the C4 PEPC clone, A3. The amount of G6P required for 50 % stimulation is higher in C4 clones (3. 7-5. 5 mM) than for C3 clones (1.0-2.5 mM). Within the C4 clones, G6P had the greatest effect on the high Km group and the amount of G6P required for 50 % stimulation was lower (3. 7-4. 2 mM) for the the higher Km group compared to the low Km group (5-5.5 mM). 

    Malate is a non-competitive inhibitor of PEPC. The percent inhibition by 32 mM malate is similar for all the enzymes from clones. However, the concentration of malate required to give 50% inhibition varied from 5 to 33 mM. Generally, the enzymes from clones (Ki ≤ 13 nM), were more sensitive to malate inhibition than those from leaves (Ki ≥ 35 nM).
    The gene products of cloned plant PEPCs had apparently under gone modifications which have affected their kinetic properties. For example, the effect of malate and G6P is less marked on the enzyme from the leaf than from the clones. The pH optimum of PEPC in leaf extract is different to the PEPC from clones. Leaf PEPC has an optimum of 7. 2-7. 3 compared to a value of 7. 5-7. 7. These differences might be attributed to differences in the expression systems and post-translational modification in E.coli and plants. 

    In conclusion, the PEPC in cDNA clones showed differences in a variety of kinetic parameters which suggest that there are intrinsic differences between them and that distinct isoforms of PEPC are present in both½ and C4 leaves. This is the first time PEPC isoforms of C4 monocots have been isolated and characterised. The phenotypic roles of these isoforms remain to be determined. These clones are ideal for structure and function studies of the PEPC protein and could potentially be used for expression studies and to assess the tissue specificity of isoform expression. 
    Date of Award1993
    Original languageEnglish

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