Transcript Enzyme Mechanisms - Illinois Institute of Technology

Cofactors, concluded
Andy Howard
Introductory Biochemistry
30 November 2010
Biochemistry: Metabolism IV
11/30/2010
Metabolism depends strongly
on cofactors
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We’ll attend to the reality that a lot of the
versatility of enzymes depends on their
incorporation of cofactors
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Cofactor topics
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Cosubstrates
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ATP and relatives
Redox
cosubstrates
Prosthetic groups
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Thioesters
Redox prosthetic
groups
Biochemistry: Metabolism IV
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Prosthetic
Groups,
concluded
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TPP
PLP
Other prosthetic
groups
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Major cosubstrates
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Facilitate group transfers, mostly small groups
Oxidation-reduction participants
Cosubstrate
ATP
S-adenosylMet
UDP-glucose
NAD,NADP
Coenzyme A
Tetrahydrofolate
Ubiquinone
Source
Function
Transfer P,Nucleotide
Methyl transfer
Glycosyl transfer
Niacin
2-electron redox
Pantothenate Acyl transfer
Folate
1Carbon transfer
Lipid-soluble e- carrier
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Major prosthetic groups
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Transfer of larger groups
One- or two-electron redox changes
Prosth.gp.
FMN, FAD
TPP
PLP
Biotin
Adenosylcobalamin
MeCobal.
Lipoamide
Retinal
Vitamin K
Source
Riboflavin
Thiamine
Pyridoxine
Biotin
Cobalamin
Function
1e- and 2e- redox transfers
2-Carbon transfers with C=O
Amino acid group transfers
Carboxylation, COO- transfer
Intramolec. rearrangements
Cobalamin
Methyl-group transfers
Transfer from TPP
Vision
Carboxylation of glu residues
Vitamin A
Vitamin K
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NAD+ and NADP+
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
Net charge isn’t really >0 ;
the + is just a reminder that the
nicotinamide ring is positively charged
Most important cosubstrates in oxidationreduction reactions in aerobic organisms
Structure courtesy of
Sergio Marchesini, U.
Brescia
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Differences between them
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The chemical difference is in the
phosphorylation of the 2’ phosphate group of
the ribose moiety
The functional difference is that NAD+ is
usually associated with catabolic reactions
and NADP+ is usually associated with
anabolic reactions
Therefore often NAD+ and NADPH are
reactants and NADH and NADP+ are products
Exceptions: photosynthesis and ETC!
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How do we get back to the
starting point?
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NADH is often oxidized back to NAD+ as
part of the electron-transport chain
NADPH is created via photosynthesis
Imbalances can be addressed via
NAD Kinase (S.Kawai et al (2005),
J.Biol.Chem. 280:39200) and NADP
phosphatase
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Reduced forms of NAD(P)
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Reduction occurs on the
nicotinamide ring
Ring is no longer netpositive
Ring is still planar but
the two hydrogens on
the para carbon are not
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NADPH
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Provides reducing power for anabolic
reactions
Often converting highly oxidized
sugar precursors into less oxidized
molecules
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FAD and FMN
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Flavin group based on riboflavin
Alternate participants in redox reactions
Prosthetic groups
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tightly but noncovalently bound to their enzymes
That protects against wasteful reoxidation of reduced
forms
FADH2 is weaker reducing agent than NADH:
when used as an energy source, it yields 1.5
ATP per oxidation, whereas NADH yields 2.5
These are capable of one-electron oxidations
and reductions
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FAD and FMN structures
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FAD has an AMP attached P to P
Structure courtesy
Paisley University
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FMN/FAD redox forms
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Two-electron version: H+ + :H- transferred
Reaction diagram courtesy of Eric
Neeno-Eckwall, Hamline University
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iClicker quiz question 1
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Based on what you have learned, would
you expect glycogen synthase to be
activated or inhibited by phosphorylation?
(a) activated
(b) inhibited
(c) neither
(d) insufficient information to tell
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iClicker quiz question 2
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What would you expect to be the
phosphate donor in the NAD kinase
reaction?
(a) free phosphate
(b) pyrophosphate
(c) ATP
(d) pyridoxal phosphate
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Thiamine Pyrophosphate
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Based on thiamine, vitamin B1
Carboxylases and oxidative
decarboxylases use this coenzyme
So do transketolases (move 2 carbons at a
time between sugars with keto groups)
Thiazolium ring is reactive center:
pKa drops from 15 in H2O to 6 in enzyme
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TPP
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Derived as in fig.17.17
We already talked about
decarboxylations of ketoacids, e.g.
pyruvate + H+ 
acetaldehyde + CO2
Formation and cleavage of
-hydroxylactones &
-hydroxyacids:
2 pyruvate + H+ 
acetolactate + CO2
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TPP reactions
pyrimidine
thiazolium
Diagram courtesy of
Oklahoma State U.
Biochemistry program
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Pyridoxal
phosphate
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PLP is prosthetic group for many aminoacid-related enzymes, particularly
transaminations
That’s how a lot of -amino acids are
synthesized from the corresponding ketoacids:
H3N+—CHR1—COO- + O=CHR2-COO- 
O=CHR1-COO- + H3N+—CHR2—COO-
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How PLP functions
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Carbonyl group of PLP bound
as a Schiff base (imine) to amino group of lysine at
active site
First step is always formation
of external aldimine; goes
through gem-diamine
intermediate to internal
aldimine
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PLP
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Remember we said it
gets used in a lot of
transaminations
We should consider its
chemistry and its other
roles in pathways
To start with: it exists in
2 tautomeric forms
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PLP:
Non-transamination reactions
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-decarboxylation:
-amino acid + H+  CO2 + H3N+-CH2-R
-decarboxylation
Others listed in fig. 17.26
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PLP intermediates
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See fig.17.27: it’s complex but important
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Biotin
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Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl
groups
… and in ATP-dependent carboxylations
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Biotin reactivity
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Covalently bound to active-site lysines to
form species called biocytin
Pyruvate carboxylase is characteristic
reaction:
Diagram courtesy
University of Virginia Biochemistry
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Tetrahydrofolate
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Primary donor of one-carbon units
(formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an
interesting drug target
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Methotrexate as cancer chemotherapeutic:
cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial:
Bacterial DHFR is somewhat different from
eucaryotic DHFR because bacteria derive DHF
from other sources; humans get it from folate
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THF structure and function
Figure courtesy
horticulture program,
Purdue
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Tetrahydrofolate variations
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-2 oxidation state:
methyl donor from N5-methyl-THF
0 oxidation state: methylene donor from
N5,N10-methylene-THF
+2 oxidation state:
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formyl (-CH=O) from N5-formyl-THF and N10formyl-THF
Formimino (-CH=NH) from N5-formimino-THF
Methenyl (-CH=) from N5,N10-methenyl-THF
See table 17.6 for specifics
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Thymidylate cycle!
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Remember that thymidine is
the rate-limiting reagent in DNA synthesis
Thymidylate derived from uridylate in a 5,10methylenetetrahydrofolate dependent
reaction:
uridylate + 5,10-meTHF 
thymidylate + dihydrofolate
Catalyzed by thymidylate synthase
Rest of cycle gets DHF reconverted into
5,10-meTHF
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The restorative reactions
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Dihydrofolate reductase (DHFR):
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DHF + NADH  THF + NAD
Enzyme is popular drug target, as suggested
Serine hydroxymethyltransferase (SHMT):
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THF + serine  5,10meTHF + glycine
This also serves as a common synthetic
pathway for creating glycine from serine
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Cobalamin
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Largest B vitamin
Structure related to heme but missing
one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements
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Catabolism of odd-chain fatty acids
Methylation of homocysteine
Reductive dehalogenation
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AdenosylCobalamin
Reactive
Co-C bond
“Missing” carbon
Diagram courtesy of
Swiss Food News
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Lipoamide
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Protein-bound form of lipoic acid
Contains five-membered disulfide ring
Covalently bound via amide to protein
lysine sidechain
Involved in swinging arm between active
sites in multienzyme complexes
Disulfide breaks, re-forms during activity
Examples: pyruvate dehydrogenase
complex, -ketoglutarate dehydrogenase
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Lipoamide 2e- reduction
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thioester starting point
Fig. Courtesy Biochem
and Biophysics
program, Rensselaer
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iClicker quiz question 3
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Which coenzyme would you expect
would be required for the reaction
oxaloacetate + glutamate 
aspartate + -ketoglutarate?
(a) ascorbate
(b) PLP
(c) thiamine pyrophosphate
(d) NAD
(e) none of the above
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iClicker question 4
A transamination is
 (a) A simple substitution of N for O
 (b) A redox reaction
 (c) Possible only at high pH
 (d) Energetically unfavorable
 (e) none of the above
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