GENERAL PROPERTIES OF ENZYMES
- enzymes are effective biological catalysts  bring reactants together in optimal orientation
- catalysts speed up reaction by stabilizing transition state between reactants and products 
lower activation energy for reaction; catalysts not consumed
- enzymes are highly specific for substrates; one enzyme can react with any of a group of
related substrate  hexokinase; others are highly specific  glucokinase
- enzymes have a high degree of reaction specificity  helps to ensure that wasteful or toxic
byproducts are not produced as can occur (which occur in non-enzymatically catalyzed
reactions, such as acetone)
1,2 MOLECULAR RECOGNITION – GENERAL ASPECTS
- proteins have the ability to specifically recognize other molecules; central to function of the
particular protein (enzyme, receptor, antibody)
- binding pockets serves as major site of interaction with specific target molecules; features:
1. ionic bonds (electrostatic interactions/salt bridges) : between acidic and basic groups
on proteins and groups of opposite charge on target molecule
2. hydrophobic interactions between non-polar groups
3. hydrogen bonds between protein and target
4. shape of binding pocket, which must accommodate target molecule so that above
interaction can occur
- enzymes must also acts as catalysts to chemically modify target (substrate); enzymes must also
possess active site where chemical modification takes place
3. PROTEASES AS AN EXAMPLE OF ENZYMES
- enzymes participate in hydrolysis of bonds; proteases catalyze hydrolysis of peptide bonds
of a protein  release of protein fragment; proteases can degrade proteins to constituent amino
acids; in other cases, proteases act to specifically process, and activate, previously inactive
form of an enzyme (zymogen)
- proteases may need help of specific amino acid side chains that are able to reversible ionize
 histidine, serine, and aspartic acid
Class of protease
Active site
Important in
Examples
Serine
Serine, histidine,
Digestion, clotting,
Trypsin,
aspartate (catalytic
tissue remodeling
chymotrypsin,
triad)
elastase, blood
coagulation,
cathepsins A,G
Aspartic
Two aspartates
Digestion, bp
Pepsin, renin,
regulation, viral
cathepsins E
pathogenesis
Cysteine
Gln-Ala-Cys-Arg
Apoptosis, lysosome
Interleukin converting
(intracellular)
enzyme, caspases 1-9,
other cathepsins
Metalloproteinases
Zn2+
Degradation of ECM
Collagenases,
stromelysins,
gelatinases
4. ZYMOGENS AND POLYPROTEIN PRECURSORS
- proteases usually synthesized in inactive form as part of large precursor containing several
proteins within it (polyprotein) or as a zymogen (proenzyme)
- both types of precursors must be proteolitically cleaved to release enzymes when they are
needed; most mammalian proteases synthesized/secreted in inactive zymogen form  activated
(via proteolysis) at the site where needed and not at site of synthesis
- mammalians also synthesize peptides as part of polyproteins; POMC is a neuropeptide
precursor that regulates the production of enkephalin, MSH, and ACTH
- activation of zymogens/polyproteins by cleavage is one-time. all or nothing event; appropriate
in viral life cycle (and in blood clotting/digestion)
- regulation of metabolic enzymes requires reversible and fine-tuned forms of control
5. HIV PROTEASE POLYPROTEIN
HIV life cycle
- examples of enzyme precursors
- production of new viral particles in an HIV-infected cell involves series of sequential events
- viral maturation relies on the cleavage of the polyprotein precursors gag and pol to smaller
functional units
-cleavage events carried out by virus’s own protease, which is made as part of the large pol
polyprotein; maturation of these proteins involves the release of active HIV protease from
pol; high concentrations of the uncleaved pol polyprotein have enough protease activity to
initiate this event and begin the cycle
- once active protease is released  it cleaves both gag and pol  releases structural proteins
required for viral assembly and reverse transcriptase, RNaseH, and integrase (needed to
begin a new cycle of infection); these protein products (except for HIV protease) are packaged
into new virus
HIV protease structure
- HIV protease is a homodimer; each subunit contains 8 beta-sheets and a single alpha-helix
- two aspartates are located in the active site  composed of two loops (one from each
subunit) locked in a fireman’s grip
- several interactions exist between the two subunits which are crucial in dimer formation
- including a group of hydrogen bond interactions in the antiparallel array formed by the Nand C- termini of both subunits (interchain H-bonds)
- intrachain H-bonds in central portion of each subunits between the N-terminus of one subunit
and the C-terminus of other
- intrachain salt bridges between active site loops and the alpha-helix of each subunit
- hydrophobic attractions between side chains that are buried in the interior of each subunit
(such as where beta strands cross each other)
- substrate binding pocket  adjacent to active site
- HIV protease recognizes a group of seven or eight consecutive amino acids in the substrate
to be cleaved
- some residues which lie close to the substrate occur in regions which appear to be some
distance away from the substrate binding pocket (however, side chains of residues project
toward pocket)
HIV substrate binding specificity (look in notes)
- HIV protease cleaves its two natural substrates, HIV gag and pol polyproteins
- HIV protease first cleaves itself out of the pol polyprotein; this is only possible when the
concentration of pol reaches high levels at the cell membrane
- because HIV protease is still part of the polyprotein when this process begins, these two sites
are thought to be favorable sites for recognition by this protease
- after HIV protease has cleaves itself out and formed mature dimers  it proceeds to cleave
the remaining two sites in the pol polyprotein as well as five sites within the gag polyprotein
- these cleavage events release individual proteins from the polyproteins which serve either as
structural elements of the virus particles, or the enzymes themselves (reverse transcriptase,
RNaseH, and integrase)
- the two autocleavage sites may be closer to the ideal recognition criteria than the other sites; a
Phe-Pro (or Tyr-Pro) sequence at position P1 and P1’, respectively, have been proposed as part
of a consensus HIV protease site
- large amount of variation in the other five positions (P4, P3, P3, P2’ and P3)  mystery as to
exact criteria for substrate recognition by HIV protease
- HIV protease is capable of specifically recognizing and cleaving gag and pol at the exact sites
required for release of HIV structural proteins, as well as itself
- HIV protease shows a preference for its natural gag and pol substrates over other proteins;
when natural gag and pol proteins are denatured so as to disrupt secondary and tertiary
structure  ability to serve as substrates is diminished; native tertiary structure of gag and
pol polyproteins play an important role in recognition by this protease (increased difficult of
designing specific inhibitors of HIV protease)
6. Clinical premise: design of successful HIV protease inhibitors
- inhibitors of HIV protease incorporate the following features:
1.) possess ability to penetrate infected cells after oral administration
2.) bind to HIV protease with high affinity; because they resemble in shape the P4-P3-P2-P1P1’-P2’-P3’ peptide that is recognized by the HIV protease (thus filling part or all of the
substrate binding site)
3.) they are not hydrolyzed by the enzyme, and thus remain to obstruct the site, thus preventing
gag and pol proteins from being processed
- HIV protease inhibitors used for HIV therapy: Saquinavir, Ritonavir, Indinavir, Nelfinavir,
Amprenavir
- due to high rate of mutations in HIV polyproteins  variants of protease arise which
possess altered binding sites that discriminate against inhibitors, while retaining ability to
cleave its natural substances
- to stall this resistance  it is common to use 2 or more different inhibitors in combination
therapy  increases time that virus requires to mutate and escape the effects of inhibitors (it
must mutate to defeat all inhibitors, not just one)
- such combination therapy has lead to a decrease in the production of viral particles in some
AIDS patients