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Unformatted text preview: CHEM 350: Introduction to Biological Chemistry
Brian Lee, Ph.D.
Ofﬁce: Neckers 146G or 324
Hours: 9:30am to 10:30am or by appointment
Textbook (required, U.S. edition only)
Fundamentals of Biochemistry, 3rd Ed., Voet, Voet & Pratt.
Study Guide (recommended)
Student Companion to Fundamentals of Biochemistry, 3rd Ed.
Tuesday 6:30 to 7:30 pm in Neckers 218
Thursday 5:00 to 6:00 pm in Neckers 410 Announcements
Undergraduate Research Opportunities
Research for credit (such as CHEM 396 or CHEM 496)
Student worker ($8.00 per hour) (http://www.siu.edu/~fao/jobs/)
Undergraduate Assistantships (http://undergraduateassistantship.siuc.edu/)
McNair Scholars Program (http://www.siu.edu/~mcnair)
REACH Awards Competition (http://www.siu.edu/~reach/) Assignments
Read Chapter 12 Enzyme Kinetics
Chapter 12 Problems
Student Companion site for Voet, Voet & Pratt
http://bcs.wiley.com/he-bcs/Books?action=index&bcsId=4274&itemId=0470129301 Third Midterm Exam, Wednesday March 28th
Tuesday 6:30 to 7:30 pm in Neckers 218
Thursday 5:00 to 6:00 pm in Neckers 410 Catalytic Mechanisms • Acid-base catalysis
• Covalent catalysis
• Metal-ion catalysis
• Electrostatic catalysis
• Proximity and orientation
• Transition state binding Lysozyme - antibacterial agent, lyses bacterial cell walls
-attacks carbohydrates in peptidoglycan
-cleaves glycosidic bond between Mur2Ac and GlcNAc
-Mur2Ac: N-acetylmuramic acid (NAM)
-GlcNAc: N-acetylglucosamine (NAG) found in tear drops
an d egg whites cleaves between
D and E Transition state binding to
the distorted half-chair
conformation of NAM
promotes cleavage of the
glycosidic bond. Isotope labeling
scissile bond Page 343 Mechanism of reaction
without enzyme using
general acid catalysis.
intermediate must be
high pKa due
Asp52 COO- stabilizes the
carbocation-oxonium ion Glu35 COOH
catalysis. Covalent Catalysis
attack on the
intermediate. Structural evidence of a covalent
intermediate using ﬂuorinated
substrate analog. C2-F slows
product release and C1-F helps
covalent intermediate formation.
noncovalent complex (yellow)
D ring distorted half chair covalent complex (green) Serine Proteases
Chymotrypsin - peptide bond cleavage on carboxyl-terminal
side of large hydrophobic side chains: tryptophan, tyrosine,
phenylalanine & methionine Covalent modiﬁcation - nucleophilic catalysis
Ser195 is highly reactive Chymotrypsin
Trp, Phe or Tyr
(also Leu or Met)
Ser, His and Asp psin lysine
speciﬁcity Evidence for an acyl-enzyme intermediate from the
“burst phase” of pre-steady state kinetics.
Chymotrypsin is capable of ester hydrolysis.
a) p-nitrophenol is rapidly formed at nearly the same
stoichiometry as the enzyme used in the reaction.
b) Then a constant “steady-state phase” of release. Chymotrypsin follows a Ping Pong Bi Bi Mechanism E-OH + pNPhAc -> E-pNPhAc -> E-Ac + pNPh
E-Ac + H2O -> E-Ac-H2O -> E-OH + Ac Enzyme activity is often dependent on pH.
The optimal pH for catalytic activity usually matches
the physiological conditions of the enzyme. pH 7.8
pH 1.6 His57 participates in catalysis Chymotrypsin
rate dependence on pH kcat dependence on pH His57-imidazole pH 8.0 KM dependence on pH
binding pocket Ile16-NH3+ Activation of Serine in the catalytic triad Catalytic triad: Asp102 -> His57 -> Ser195
-hydrophobic pocket His57 acts as a general base catalyst
and activates Ser195 hydroxyl to
attack the carbonyl carbon. Asp102-His57 low barrier H-bond raises pKa of His57 For histidine to act as a
base catalyst, it must be
or ... pKa 14.5
pKa His57 13 Ser195
pKa = 6.04
... the ﬁrst pKa must be
much higher than 6.04. Problem: How do you
remove a proton from
Histidine (pKa 14.5) with
an Aspartate (pKa 3.9) pKa 14.5
His57 pKa = 3.90
Asp102 pKa = 6.04
This cannot be done in a
normal acid-base reaction. Hydrogen bonding
between histidine and
aspartate will increase
the pKa of histidine... Asp102 His57 pKa = ??? ...but not enough to
abstract a proton
from serine. Ser195
Asp102 His57 pKa = ???
If the hydrogen bond is stronger,
then the pKa will increase enough
to allow histidine to act as a base. What is a Low-Barrier
Hydrogen Bond (LBHB)?
Strong than normal.
about 60 kJ/mol
versus 20 kJ/mol
If the LBHB forms in
response to substrate binding, then the increase binding
energy directly contributes to GB and the reduction in G .
Note: Aps102 has a lower (or equal) pKa than His57, and
normally would not be able to accept a proton from His57.
The LBHB is the “best” explanation of how Asp102 helps
to partially abstract a proton from His57 Oxyanion hole stabilizes negatively charged carbonyl
oxygen in the tetrahedral intermediate through H-bonds. acyl-enzyme intermediate His57 can now act as a
general acid catalyst,
donating a proton to
the new amino group.
The carbonyl double bond
reforms as the peptide
bond is broken.
First product is
Ping Pong mechanism The acyl-enzyme intermediate
is a long lived covalent complex.
Waiting for water… Water binds in the same location
as the departed amino group,
stabilized by H-bonding to His57. Reversal of previous reactions
to release second product and
regenerate the enzyme. His57 now acts as a general
base and removes a proton
from water to create a
nucleophilic hydroxide ion. The tetrahedral intermediate
now involves the bound
water molecule. Collapse of the short-lived intermediate His57 acts as a general acid to
donate a proton back to Ser195.
The second product is released and the
enzyme has returned to it original state. Evidence for the tetrahedral intermediate:
BPTI prevents trypsin from digesting the pancrease
and binds to trypsin with 0.1 x 10-12 M afﬁnity. X-ray structure of BPTI bound to trypsin shows the
tetrahedral intermediate prior to breaking the peptide bond The structure of another serine protease, elastase,
shows the second tetrahedral intermediate with water.
pH 5.0 structure
with water bound pH 9.0 structure of
tetrahedral intermediate water Chymotrypsin (EC 126.96.36.199)
Classiﬁcation - 3) Hydrolase: hydrolysis reaction
(transfer of functional groups to water)
-acid-base catalysis (His57)
-covalent catalysis (Ser195)
-electrostatic catalysis (oxyanion hole)
-transition state binding More serine protease enzymes Trypsin is closely related to chymotrypsin
The substrate binding pocket of trypsin has an
Aspartic acid residue at the bottom of the pocket
Speciﬁcity changes from large hydrophobic
to large basic residues: Lys and Arg. Unrelated serine proteases have evolved independently
and contain the same catalytic triad residues: Asp, His, Ser Other Classes of Proteases
carboxypeptidase A Zymogens – inactive precursors of enzymes.
Digestive enzymes are ﬁrst synthesized as zymogens
to prevent digestion of tissue and organs. As trypsinogen leaves
the pancrease, it is
initially activated by
Trypsin can also
for autocatalytic activation The ﬁbrin network in blood
clots is formed through the
cleavage of ﬁbrinogen by
Thrombin is activated by
a cascade of clotting
factors. Many of the
clotting factors are
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