CELLULAR BIOCHEMISTRY

1
CELLULAR BIOCHEMISTRY PROTEINS AND
ENZYMES LECTURE 7 ENZYME-SUBSTRATE COMPLEXES AND
THE ACTIVE SITE
2
ENZYME-SUBSTRATE COMPLEXES AND THE ACTIVE SITE
ENZYME-SUBSTRATE COMPLEXES The E-S complex is a
central feature of the Michaelis-Menten approach
to enzyme kinetics. E-S complexes are transient
(and usually very unstable). Some direct evidence
for them comes from spectroscopic studies and
other approaches 1. Spectroscopic studies
where enzyme and ES complex have different
spectroscopic properties - e.g. peroxidase, which
catalyses Pyrogallol H2O2 ? oxidized
pyrogallol H2O
3
EVIDENCE FOR ENZYME-SUBSTRATE COMPLEXES (CONT)
2. X-ray studies on complexes between enzyme and
'sluggish' substrate or inhibitor - e.g.
Lysozyme (see next lecture). 3. Trapping of
covalent intermediates (e.g. chymotrypsin). 4.
Identification of covalently modified form of an
enzyme formed during the course of reaction (e.g.
phosphoglucomutase - see below) 5. Binding of
one substrate to enzyme in a 2 substrate reaction
(e.g. dehydrogenases) so E-S complexes are
formed they are specific, stoichiometric and in
at least some cases recognisably covalent N.B.
the enzyme really is involved in the reaction.
4
THE ACTIVE SITE
The Lock-key hypothesis (Emil Fischer) proposes
that the substrate fits into the active site as a
key does into a lock. The Induced fit theory
(Koshland) on the other hand suggests that in
some cases the conformation of the active site
changes as the substrate arrives, increasing the
goodness of fit between the two.
5
LOCK-KEY
INDUCED FIT
6
HOW CAN WE INVESTIGATE THE ACTIVE SITE AND
ASSOCIATED REACTION MECHANISMS?
1. Use kinetic studies. 2. Look at the
specificity of substrates (and inhibitors) whose
shape may complement that of the active site. 3.
Identify enzyme-substrate complexes directly
e.g., phosphoglucomutase Location of tightly
bound coenzymes or prosthetic groups can also
give important clues. 4. Use reagents that
covalently modify the active site (irreversible
inhibitors) e.g. modification of active site
Ser on trypsin, chymotrypsin etc. by
Diisopropylphosphofluoridate (DIPF)
Also Affinity labelling and general chemical
modification. 5. X-ray analysis -
identification of active site and complexes with
it ( as for lysozyme etc.) 6. Protein
engineering

7
CATALYSIS AND SPECIFICITY REVISITED
CATALYSIS Enzymes speed up reactions 106 - 1020
fold. Usually more active than non-enzyme
catalysts. Some enzymes may reach catalytic
perfection

• SPECIFICITY
• Examples
• amino acid activating enzymes (discriminate
  between very similar amino acids with
  fidelity better than 1 part in 5000)
• Stereospecificity (discrimination between D and L
  isomers of a substrate)
• Phosphoglucomutase (discriminates between the
  different -OH groups on a substrate)

BUT some enzymes show low specificity - e.g.
alkaline phosphatase (which hydrolyses phosphate
groups from many different substrates) Specificit
y is determined by binding site and orientation
of catalytic groups. It implies specific 3D
interaction between enzyme and substrate.
Another aspect of specificity is lack of side
reactions (specificity with regard to products).
8
REVERSIBLE ENZYME INHIBITORS
Some inhibitors (e.g. DIPF) are irreversible.
They bind covalently to the enzyme and
essentially 'kill' it. Others are reversible -
they bind non-covalently, and on removal activity
is recovered.

• Two major types of reversible inhibitor
• Competitive inhibitors - bind to the active site,
  like the substrate, but are not converted to
  products. They block access of substrate to the
  active site, but increased substrate
  concentration can overcome the inhibition - there
  is competition between substrate and inhibitor.
  An example is succinate dehydrogenase
• Non-competitive inhibitors - usually bind away
  from the active site, and do not compete for
  substrate.

• allosteric inhibitors, sometimes considered a
  subset of non-competitive inhibitors will be
  discussed in Lecture 9

9
PHOSPHOGLUCOMUTASE
1
1
10
Diisopropylphosphofluoridate
11
SERINE PROTEASES - ACTIVE SITES
TRYPSIN
Glu-Gly-Asp-Ser-Gly-Gly-Pro-Val CHYMOTRYPSIN
Met-Gly-Asp-Ser-Gly-Gly-Pro-Leu ELASTASE
Gln-Gly-Asp-Ser-Gly-Gly-Pro-Leu THROMBIN
Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe
Ser OH CH2
-NH-CH-CO-
12
ENZYME TURNOVER NUMBERS (kcat)
kcat (per sec) Carbonic anhydrase 600,000 A
cetyl cholinesterase 25,000 Penicillinase
2,000 Lactate dehydrogenase
1,000 Chymotrypsin 100 DNA
polymerase 15 Lysozyme 0.5
molecules of substrate converted/molecule of
enzyme/sec
13
COMPETITIVE INHIBITION OF SUCCINATE DEHYDROGENASE