Table 211 Activity of pyruvate carboxylase enzyme PYC isolat

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

Following purification of the PYC enzyme by avidin-Sepharose affinity chromatography, the investigators carried out several experiments to characterize the 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 protein through a calibrated gel filtration column. The results from the gel filtration column indicated that the PYC enzyme had a molecular weight of 540 kilo-daltons. Use this information to determine the structure of the PYC enzyme. 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^2+ ions as a control. In addition, the dependence of the enzyme on these various metabolites was tested by replacing them with similar compounds. The activity of the enzyme in the presence of these various effectors is shown in Table 21.1. Explain why PYC had no activity in the presence of avidin. Is PYC dependent on ATP for activity? Can other nucleotides substitute for ATP? What is the effect if other nucleotides are added to the assay mixture in addition to ATP? What is the effect of other tricarboxylic acid metabolites on PYC activity in the methanogen? What ion or ions are required for PYC activity? 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?

Solution

7) a. Pyruvate carboxylase enzymes are dependent on the protien bound biotin, as biotin forms the key region for the active site of PYC (Protein bound enzyme), thus in presence of avidin, the rate of activity of PYC was inhibited as avidin binded to biotin. This would not have been the case if excess amount of biotin would have been incubated in the culture.

b. The PYC(pyruvate carboxylase enzyme) was strictly dependent on ATP, no other replacements

c. No, there has been no activity of PYC with otherconstituents in TCA Tricarboxylic acid cycle, neither with Acetyl Co A nor with aspartate.

d. Mg2+, Co2+, Mn2+ had effect on activity of enzyme