Study Guide
BioChem 330 - Course Outline 2011 Part 3
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University:
Amherst College -
Course:
CHEM 330 | Biochemical Principles of Life at the Molecular Level Academic year:
2011
-
Views:
157
Pages:
18
Author:
Kaylyn Howard
Gly 4 Aldolase
Title Fructose 1,6-Bisphosphate
Aldolase From Rabbit Muscle
Classification Lyase Compound
Mol_Id: 1; Molecule: Aldolase;
Chain: A, B, C, D; Ec: 4.1.2.13;
Biological_Unit: Tetramer
Exp. Method X-ray Diffraction
1ADO
Active site with Lys41 shown in blue, substrate
analogue spacefilled and cpk colored Gly 4 Aldolase 1ADO
Active site Tyr 301 shown spacefilled in green Gly 5 Triose Phosphate Isomerase TIM
H
O
T ri ose Isom era se
H
OH
H2 C
O
H
5.3.1.1
HO
H
O
O
P
Glyceraldehyde3-phosphate
O
H2 C
O
O
O
P
O
O
Dihydroxyacetone
phosphate
DGo’ ketone to aldehyde = +7.5 kJ/mole prod/react =1/20 Gly 5 TIM Mechanism
O
O
O
O
P
O
CH2
O
H
O
O
O
CH2
H
P
HB
Triose
Isomerase
OH
O
C
Glu-165
O
H
O
Triose
Isomerase
B
CH2
H
OH
HB
H
O
O
O
O
P
O
H
H
O
O
C
H
O
O
Glu-165
O
C
Glu-165
• Enol formation requires both protonation and
deprotonation
• Two groups involved in proton shuttles: Glu165 and His-95. Gly 5 TIM Kinetics for DHAP
• kcat = 2.0 x 105 s-1 (ΔGtt = 10.4 kcal/mole)
– modest catalysis
• Km = 8.7 x 10-4 M
– enzyme binds just well enough (0.87 mM)
• kcat/Km = 2.4 x 108 M-1s-1
– virtually the highest possible for bimolecular process of E +
DHAP = E + GAP, TIM is the perfect enzyme
• Close to saturation?
– [S] = [DHAP] = 50 uM
– Km = 870 uM
– Km/S = 17/1 enzyme not working at Vmax, can go faster if
necessary Gly 5 TIM structure
• Enzyme Motion
– When the enzyme binds its substrate the
loop closes over the active site, shielding
the substrate from water. The loop
appears to close as a rigid lid, stabilized by
internal hydrogen bonds. Its motion
involves movements of alpha carbons of
up to 8 A. The closure involves the filling
of a cavity near the base of the helix to
which the loop is connected and the
formation of new hydrogen bonds and
contacts.
– Structures 2YPI , 3TIM , 6TIM Gly 5 TIM Structure 2YPI
Crystallographic Analysis Of
The Complex Between
Triosephosphate Isomerase
and 2-Phosphoglycolate At
2.5 Å
Classification
Isomerase (Intramolecular
Oxidoreductse)
Compound Triose
Phosphate Isomerase (TIM)
(E.C. 5.3.1.1) Complex With 2Phosphoglycolic Acid
Exp. Method X-ray
Diffraction Gly 5 TIM Structure 2YPI
Glu 165 that acts as a base to
abstract a proton is shown in
blue at right
His95 that participates in acid/base
mechanism shown in yellow at left. Phase II
2GAP + 2NAD+ + 4 ADP+ 4 HPO4-2
---> 2 pyruvate + 2 NADH + 4 ATP + 6 H2O
Cashing In, here we see a production of
2 NADH and when we subtract the 2 ATP
invested from 4 ATP above, net 2 ATP
Note that reaction above isn’t charge or
mass balanced, and protons aren’t
accounted for directly, some reactions will
generate.
6 waters produced, 2 come from substrate
in gly 9 and 4 come from gly 7 and gly 10
(ADP-4 + HPO4-2= ATP-6 + H2O+H+) Gly 6: Glyceraldehyde-3-Phosphate
Dehydrogenase:
1.2.1.12
O
[HPO4]-2 + H
H
Gl ycera ld ehyde -3 -p hosp hate O
de hydrogen ase
OH
H2 C
O
O
Glyceraldehyde3-phosphate
O
P
O
O
P
O
H
O
OH
H2 C
O
NAD+
NADH
O
1,3-diphosphoglycerate
+ H+
O
P
O
O
2
Free Energy
• Note: overall oxidation
• Exergonic oxidation of
aldehyde drives reaction
• Cofactor: NAD+
0
-2
-4
-6
-8
-10
-12
Start
Hexokinase
PGI PFK
A ldolase
TIM
G-3-P DH
PGKPGM
EnolasePK
DGo’ = +6.3 kJ/mole Gly 6:
Mechanism
*C149 attacks GAP,
H176 grabs proton
*thiohemiacetal
intermediate
* hydride shift to NAD+
GAP oxidized
* second acylthioester
intermediate
* NADH release is rate
limiting
*second NAD+ binds
* Pi is 2nd substrate
*1,3 BPG released
This step is where the
substrate is oxidized!!
Base is
H176
Deprotonation of phosphate
yields H+ in product
Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 6: Saturation? Km and [S]
GAP + NAD+ + Pi = 1,3 DPG + NADH
Km
[S]
Km/[S]
GAP
70 uM
3 uM
23
less than ½ Vmax
NAD+
46 uM
600 uM
0.08
sat’d w NAD+
Pi
high
2,000 uM
>10
weak binding
•Km/S<< 1 predicts that gly6 is saturated w NAD+ and not sensitive to
changes in it, or controlled by its concentrations
•GAP and Pi both have Km/S >1, both are >10 for very different reasons.
GAP has good binding but a low concentration of substrate. Pi has
horrible binding, but a high concentration of substrate. Since the binding
won’t change, we say that reaction is controlled by GAP because small
changes in its concentration will dramatically alter the rate. Gly 6:Structure 1GAD.
* Dimer structure
from e.coli
* NAD+ binding in
Rossman fold
* Pos. Cooperativity
Hill 2.3 Gly 7: Phosphoglycerate Kinase
First ATP Generation
2.7.2.3
O
O
O
O
P
Phosphoglycerate
Ki nase
O
H
OH
H2 C
O
O
P
OH
O
O
P
O
O
3-Phosphoglycerate
2
0
Free Energy
• Very exergonic , ATP
generated
• Only monomeric enzyme on
pathway
• Closely associated with Gly6
-18.5 kJ/mole
H
ADP ATP
1,3-diphosphoglycerate
DGo’ =
O
H2 C
O
O
O
-2
-4
-6
-8
-10
-12
Start
Hexokinase
PGI PFK
A ldolase
TIM
G-3-P DH
PGKPGM
EnolasePK Gly 7: Structure 16PK from Trypanosoma brucei
* ADP binding site looks
like NADH binding site,
here crystallized with
NADH analogue
* ADP binds tightly,
more like cofactor, Km/S
= 0.05
* 1,3 DPG Km/S = 9 Gly 8: Phosphoglycerate Mutase
O
O
H
OH
H2 C
O
O
P
Phosphogly cerate
Mutase
O
2.7.5.3
H
O
O
O
H2 C
O
OH
P
O
O
O
3-Phosphoglycerate
2-Phosphoglycerate
DGo’ = +4.4 kJ/mole
2
Free Energy
0
-2
-4
-6
-8
-10
-12
Start
Hexokinase
PGI PFK
A ldolase
TIM
G-3-P DH
PGKPGM
EnolasePK Gly 8: PGM Mechanism
*Base (Lys?) interacts with
carboxylic acid of 3PG for
binding
* Active enzyme phosphorylated by 2,3-DPG
*His His duo at active site
*Km varies from 240 uM in
brain to 5000 uM in muscle.
(nature of base?)
*[S] 3PG 40-60 uM
*Km/S = 6-80, E not sat’d
with S, room to speed up.
Lys
Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 8: 3PGM structure from
Schizosaccharomyces pombe
• Gly 8 has a relatively
simple structure with a
beta twist and helical
unit, 3PG binds at
interface.
• Two his 4 Å apart shown
in blue wireframe
• His adjacent to C2 has Pi,
His adjacent to C3
extracts Pi from C3 while
OH on C2 extracts Pi from
Pi-His
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