Tost Mwt Stds, KD 272 132 95 || Non-denaturing Denaturing 11 - 66 - 45 14

Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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The enzyme pyruvate carboxylase is discovered in a bacterium that was thought not to contain it; in this case study, you’ll see how researchers study and characterize the enzyme, and, ultimately, show how it fits into a metabolic pathway.

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 the figure to the right. In addition, they ran the protein through a calibrated gel filtration column, the results of which indicated that the PYC enzyme had a molecular weight of 540 kiloDaltons. In the figure, the rightmost column are where molecular weight standards of various sizes would occur if they’d been on the gels.

The catalytic properties of the PYC enzyme were assessed following purification. The activity of the enzyme was assayed in the presence of ATP, pyruvate, bicarbonate and Mg2+ ions (these latter ions were the control substance). 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 molecules is shown in the table.

Part A} The amino acid 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? Would this happen if the amino acid residue were not lysine?

a
10 Mwt Stds, KD
272
132
95
- 66
- 45
14
3
Non-denaturing Denaturing
Transcribed Image Text:a 10 Mwt Stds, KD 272 132 95 - 66 - 45 14 3 Non-denaturing Denaturing
Effectors
Control
Avidin
Alternate nucleotides replacing ATP of ag
AMP
ADP
CTP
GTP
ITP
UTP
Nucleotides in addition to ATP
AMP le letts onl
ADP
CTP
GTP
ITP
as orow orlysne sadyxodis
UTP disid tavura ITA 10 song
Tricarboxylic acid cycle-related compounds
Acetyl-CoA
Aspartate
Glutamate
a-ketoglutarate
Divalent cations replacing Mg²+
Mn²+
Co²+
Zn²+
Activity (% of control)
100
0
0
alog goh oob-non bas
noitibhs.IS ougil di awode o
0
wil logodilo a riguordt niston
od 0
-om a bo
104
73
106
94
80 yg sdi to zainoqong
105
84
91
95
73
17
46
0
T
D
Transcribed Image Text:Effectors Control Avidin Alternate nucleotides replacing ATP of ag AMP ADP CTP GTP ITP UTP Nucleotides in addition to ATP AMP le letts onl ADP CTP GTP ITP as orow orlysne sadyxodis UTP disid tavura ITA 10 song Tricarboxylic acid cycle-related compounds Acetyl-CoA Aspartate Glutamate a-ketoglutarate Divalent cations replacing Mg²+ Mn²+ Co²+ Zn²+ Activity (% of control) 100 0 0 alog goh oob-non bas noitibhs.IS ougil di awode o 0 wil logodilo a riguordt niston od 0 -om a bo 104 73 106 94 80 yg sdi to zainoqong 105 84 91 95 73 17 46 0 T D
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