Table 21.1: Activity of pyruvate carboxylase enzyme (PYC) isolated from M. therm
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Question
Table 21.1: Activity of pyruvate carboxylase enzyme (PYC) isolated from M. thermo- autotrophicum in the presence of various effectors (based on Mukhopadhyay, et al., 1998).
Effector
Activity (% of control)
Control
100
Avidin
0
Alternate nucleotides replacing ATP
0
AMP
ADP
0
CTP
0
GTP
0
ITP
0
UTP
0
Nucleotides in addition to ATP
104
AMP
ADP
73
CTP
106
GTP
94
ITP
80
UTP
105
Tricarboxylic acid cycle-related compounds
84
Acetyl-CoA
Aspartate
91
Glutamate
95
a-ketoglutarate
73
Divalent cations replacing Mg2+
17
Mn2+
Co2+
46
Zn2+
0
Effector
Activity (% of control)
Control
100
Avidin
0
Alternate nucleotides replacing ATP
0
AMP
ADP
0
CTP
0
GTP
0
ITP
0
UTP
0
Nucleotides in addition to ATP
104
AMP
ADP
73
CTP
106
GTP
94
ITP
80
UTP
105
Tricarboxylic acid cycle-related compounds
84
Acetyl-CoA
Aspartate
91
Glutamate
95
a-ketoglutarate
73
Divalent cations replacing Mg2+
17
Mn2+
Co2+
46
Zn2+
0
Mwt Stds, kD 272 6. Following purification of the PYC by avidin- enzyme Sepharose affinity chromatography, the investigators 132 carried out several experiments to characterize the 95 enzyme. First they ran samples of the enzyme on denaturing and non-denaturing gels. The results are shown in Figure 21.1. In addition, they ran the pro- tein through a calibrated gel filtration column. The 14 results from the gel filtration column indicated that the PYC enzyme had a molecular weight of 540 kilo- Non-denaturing Den daltons. Use this information to determine the struc- Figure 21.1: Gel electrophoresis of pyruvate ture of the PYC enzyme carboxylase purified from M thermoautotrophicum (Based on Mukhopadhyay, et al., 1998.) 7. The catalytic properties of the pyruvate carboxylase enzyme were assessed following purification. The activity of the enzyme was assayed in the presence of ATP, pyruvate, bicarbonate, and Mg ions as a In addition, the dependence of the enzyme on the various metabolites was tested by replacing them with similar compounds. The activity of the enzyme in the presence of the various is shown in Table 21.1 a. Explain why PYC no activity in the presence of avidin. ATP? What is the b. Is PYC dependent on ATP for activity? Can other nucleotides substitute for c. effect if other nucleotides are added to the assay mixture in addition to ATP? methanogen? What is the effect of other tricarboxylic acid metabolites on PYC activity in the d. What ion or ions are required for PYCactivity? 8. The sequence of PYC from the methanogen was compared to other pyruvate carboxylase enzymes and it was discovered that the lysine at position 534 is strictly conserved. Why is this the case?Explanation / Answer
Q6) The two bands signifies that the protein is made up of two subunits.
1) suppose A with near about 50 kDa
2)suppose B with 70-72 kDa
Now, the analysis of the tabulated data indicates the presence of the following subunits:
a) one must have the ATP binding site, probably carboxylase (nonbiotinylated)
b) other subunit containing keto acids and metal ion binding sites (is carboxyl transferase )
Q7) a. because of dependence of pyruvate carboxylase activity on protein-bound biotin
b. the activity is ATP dependent looking at the data. no, they cannot replace ATP but the activity would be slightly augmented / supplemented on addition of AMP, CTP, UTP nucleotides whereas, the addition of other nucleotide seems to have inhibited the activity
c.Tricarboxylic acid related compounds seems to have only reduced / iinhibited the activity. slightly inhibition by aspartate and glutamate and comparatively higher by alpha-ketoglutarate.
d. Mg2+ are required for the activity as the activity was reduced on replacement of this ion by other ions.
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