Biochemistry Ch 8 112-133
Enzymes as Catalysts
Enzymes are proteins that act as catalysts to increase rate of chemical reactions
1.) Binding of substrate: E + S  ES
2.) Conversion of bound substrate to bound product: ES  EP
3.) Release of product: EP  E + P
-Enzyme participates in making and breaking of bonds required for product formation, releases the
products and returns to its original state
-Enzymes do not make new reactions, they make them occur faster
Specificity – the ability of an enzyme to distinguish and select just one substrate for reaction
-Most tissues in body are adversely affected by alcohol, such as liver, pancreas, heart, CNS, and fetus
-many effects of alcohol are as a direct result of ethanol, but others relate to its metabolism
-Organophosphorous compounds such as Malathion and parathion are insecticides.
-ingestion may result in respiratory failure, death
-Glucokinase converts Glucose to Glucose-6-phosphate in the presence of ATP to form G6P + ATP
The Active Site – Enzyme substrate complex has to form in order to catalyze a reaction.
-Active site is usually a cleft in the enzyme formed from the polypeptide chains
-Active site also contains functional groups that are involved in the reaction
-Activated substrates and enzyme form a transition-state complex, an unstable high-energy
complex
-additional bonds help stabilize transition-state complex
-transition-state complex dissociates to form products
Substrate-Binding Sites – Enzyme specificity is the ability to bind one substrate, results from 3
dimensional arrangement of specific amino acid molecules, 2 theories for enzyme-substrate binding:
1. Lock and Key Model – active site contains residues specific for that substrate and binds it
through multiple interactions. Amino acids can come from multiple parts of the protein that has
been folded
2. Induced-fit Model – As a substrate binds, the enzyme undergoes a conformational change that
repositions the side-chains of amino acids in the active site and increases the number of binding
interactions. It is not a rigid lock, but is able to conform to the structure of the substrate
-Glucose is held together in glucokinase by multiple hydrogen bonds between hydroxyl group and polar
amino acids from the enzyme.
-Glucokinase cannot phosphorylate a galactose
Transition-State Complex – for a reaction to occur, substrates need to be activated
-highest energy level on a plot of energy and reaction progress is the transition state.
-in some reactions, transition state is when bonds of the substrate are maximally strained, in others the
electronic configuration of substrate is strained
-Activation energy – difference in energy between the substrate and the transition-state complex
Overall rate of reaction – number of molecules acquiring activation energy necessary to form the
transition-state complex
-Enzymes decrease the activation energy
-Once transition state is formed, it can collapse back to substrate and enzyme or decompose to products
-Transition-state complex binds more tightly to enzyme, and therefore other compounds that resemble
it would be potent inhibitors of the enzyme
-A drug developed as a transition-state analog would be highly specific for the enzyme it is
designed to inhibit
-Abzymes – engineering antibodies to have a particular enzyme’s active site on their variable regions,
they can act as artificial enzymes
-have been developed against analogs of transition-state complex for cocaine-esterase, enzyme
that degrades cocaine in the body
-can decrease dependence in individuals with monthly injections
Catalytic Mechanism of Chymotrypsin – chymptrypsin is a digestive enzyme released into the intestine
that catalyzes hydrolysis of peptide bonds in denatured proteins
-member of the serine-protease superfamily, which use serine in active site to form covalent
intermediate during proteolysis
-OH from H2O is added to carbonyl of peptide bond, and H+ is added to the nitrogen
-Bond that is cleaved is named the Scissile Bond
1. Reaction without chymotrypsin – without enzyme, -OH of water attacks the carbonyl carbon, which
carries a partial positive charge. The unstable oxyanion tetrahedral transition-state complex is formed
in which the oxygen atom carries a full negative charge.
-reaction is slow because there are not enough –OH molecules in H2O to form transition-state
complex and too few –OH colliding in the right orientation
2. Catalytic Strategies in Chymotrypsin reaction – the same oxyanion intermediate is formed by using
hydroxyl group of a serine residue for the attack instead of a free hydroxyl anion
-rate of reaction is faster because the functional groups in chymotrypsin stabilize the oxyanion
transition-state complex, form covalent intermediates, and destabilize the leaving group. The
reaction occurs in two stages:
a. Cleavage of peptide bond in denatured substrate protein and formation of covalent
acyl-enzyme intermediate
b. Hydrolysis of acyl-enzyme intermediate to release remaining portion of substrate
Specific Binding to Chymotrypsin – the enzyme catalyzes hydrolysis of peptide bond on the carbonyl
side of phenylalanine, tyrosine, and tryptophan
-substrate protein must be denatured to fit into the binding site and held together by glycines
Formation of Acyl-Enzyme Intermediate in Chymotrypsin –
-in the first stage of the reaction, the peptide bond of the denatured protein is cleaved as an active site
serine hydroxyl group attacks carbonyl carbon of the scissile bond
-an active site histidine acts as a base and abstracts a proton from the serine hydroxyl, and is stabilized
by a nearby aspartate
-Aspartate-Histidine-Serine combination is known as the catalytic triad
-increases the reaction rate similar to increasing the number of free –OH ions in solution
-stabilization of transition state complex lowers its energy level and increases the number of molecules
that reach this energy level
-Serine of the active site forms a full covalent bond with the carbon of the carbonyl group as a peptide is
cleaved
-formation of a covalent intermediate is a strategy of enzymes that can use either serine or
cysteine residues
-covalent intermediate is subsequently hydrolyzed
-Dissociating products of an enzyme-catalyzed reaction are often “destabilized” by some degree of
charge repulsion in active site, in the case of chymotrypsin this is an active site Histidine.
Hydrolysis of Acyl-Chymotrypsin Intermediate – active site histidine activates H2O to form OH- for
nucleophilic attack, resulting in a second oxyanion transition-state complex. When the histidine adds
the proton to serine, the reaction is complete and the product dissociates
-Pepsin in the stomach uses asparate residues in its active site for catalysis of peptide-bond instead of
histidine like chymotrypsin because histidine with a pKa of 6.0 would be protonated at a low pH and
could not extract a proton from a nucleophile
Functional Groups in Catalysis – proximity and orientation are intrinsic features of substrate binding
-a great variety occurs in functional groups in different enzymes to carry out catalysis
-some enzymes like chymotrypsin rely only on amino acids in the active site
-other enzymes hire cofactors (nonprotein compounds that participate in catalysis) to provide a
functional group of the right properties.
-Divided into coenzymes, metal ions, and metallocoenzymes
Functional groups on Amino Acid Side Chains – almost all polar amino acids participate directly in
catalysis in one or more enzymes
Coenzymes in Catalysis – complex nonprotein organic molecules that participate in catalysis by
providing functional groups, usually synthesized from vitamins
-since vitamins function as coenzymes, symptoms of vitamin deficiencies reflect the loss of specific
enzyme activities that depend on the coenzyme from the vitamin
-drugs that inhibit synthesis of coenzyme cause functional deficiency, whereas inadequate
intake is called dietary deficiency
-ethanol is an antivitamin that decreases cellular content of almost every coenzyme
-Coenzymes can be divided into two classes:
1. Activation-Transfer Coenzymes – participate directly in catalysis by forming a covalent
bond with a portion of the substrate, which is then activated.
-the portion of coenzyme that binds substrate is its functional group, and a
separate one binds tightly to the enzyme
-Thiamine pyrophosphate is synthesized from the vitamin thiamine by addition of
pyrophosphate, which acts to chelate Mg2+ ions to bind to enzyme.
-forms covalent bond with substrate keo group while cleaving the adjacent
carbon-carbon bond
-alcoholics develop thiamine deficiencies because alcohol inhibits transport of
thiamine through intestinal mucosal cells
-Coenzyme A (CoA) synthesized from vitamin pantothenate, binds enzyme using an
adenosine 3’, 5’-biphosphate and a sulfhydryl functional group that attacks carbonyl
groups and forms acyl thioesters
-CoA is transformed into products that dissociate away from enzyme -> reduced
to NADH
-Biotin does not contain phosphate group, but is bonded to a lysine in carboxylase
enzymes
-Function group is a nitrogen atom that covalently binds CO2 in an energyrequiring reaction – biotin only functions in carboxylation reactions
-Pyridoxal phosphate synthesized from vitamin pyridoxine (Vitamin B6) – has a reactive
aldehyde group to catalyze reactions – ring nitrogen withdraws electrons from a bound
amino acid, resulting in cleavage of that bond
-All of these activation-transfer enzymes have 3 things in common: specific chemical group that
binds to enzyme, a specific function group for catalysis, and dependence on enzyme for
additional specificity to substrate
2. Oxidation-Reduction Coenzymes – correspond to coenzymes involved in oxidationreduction reactions by enzymes called oxidoreductases
-oxidation is the loss of electrons, addition of O or loss of H atom
-reduction is the gain of electrons, which is a gain of an H atom or loss of an O
-Coenzymes such as NAD+ and FAD can transfer electrons together with hydrogen, can
generate ATP from oxidation of fuels
-Vitamin E and Vitamin C are redox coenzymes that can act as antioxidants and protect
against oxygen free radical injury
-a set of enzymes known as dehydrogenases because they transfer a hydrogen from a
substrate to an electron-accepting coenzyme such as NAD+
-do not form covalent bonds with substrate
-has a unique functional group that accepts and donates electrons
Lactate Dehydrogenase – catalyzes transfer of electrons from lactate to NAD+
-NAD+ is synthesized from the vitamin niacin and from ATP. ADP portion of
molecule binds enzyme and causes conformational changes to the enzyme.
Functional group of NAD+ is the carbon on nicotinamide ring opposite the
positively charged nitrogen
-this carbon acceptes hydride ion transferred from a specific carbon on
substrate, then –OH from substrate dissociates and a keto group C=O is formed
-Most ingested ethanol is oxidized to acetaldehyde in liver by alcohol dehydrogenase (ADH)
Ethanol + NAD+  acetaldehyde + NADH + H+
-In active site of ADH, the activated serine pulls proton off the ethanol leaving negative charge
on oxygen that is stabilized by zinc
Metal Ions in Catalysis – metal ions with their positive charge can help catalysis by acting as
electrophiles, and can help bind substrates or coenzymes to enzymes.
-Mg2+ helps bind negatively charged phosphate groups of thiamine pyrophosphate to anionic or basic
amino acids in the enzyme
-ATP is usually bound to enzyme via Mg2+ chelation
-Cofactors can also play a structural role in enzymes, binding different regions of enzyme together to
form tertiary structure
Optimal pH and Temperature – an increase in enzyme activity is noted as you go from acidic pH to
physiological, and a decrease from physiological to basic
-shape of curve usually reflects ionization of specific functional groups in active site
-loss of activity on the basic side usually reflects the inappropriate ionization of amino acid residues in
enzyme.
-most human enzymes function at 37 degrees Celsius, higher would denature the protein
Mechanism Based Inhibitors – Inhibitors are compounds that decrease the rate of an enzymatic
reaction, can mimic or participate in an intermediate step of catalysis, includes transition state-analogs
and compounds that bind irreversibly to functional groups in active site
1. Covalent Inhibitors – form covalent bonds with functional groups in active site, which far more
likely to be targeted by dugs and toxins than other amino acids
-diisoproprylphophofluoridate (DFP) is a lethal compound that used to be a nerve gas
and insecticide like malathion and parathion.
-forms covalent intermediate in active site of acetylcholinesterase, preventing
the enzyme from degrading acetylcholine, it is irreversible
-DFP also inhibits many other enzymes that use serine for hydrolytic cleavage
2. Transition State Analogs and Compounds that Resemble Intermediate Stages of Reaction – TS
analogs are extremely potent as inhibitors of enzymes, can also be referred to as substrate
analogs
a. Penicillin – antibiotic that is a transition-state analog that binds tightly to glycopeptidyl
transferase, an enzyme required by bacteria for cell wall synthesis. The enzyme
catalyzes a reaction with penicillin that covalently attaches it to its own active site serine
i. Sometimes called Suicide Inhibitors (irreversible binding)
b. Allopurinol – drug used to treat gout decreases urate production by inhibiting xanthine
oxidase. This enzyme commits suicide by converting the drug to a transition-state
analog
i. Xanthine oxidase normally oxidizes hypoxanthine to xanthine and xanthine to
uric acid in purine degradation.
ii. Xanthine oxidase oxidizes allopurinol to oxypurinol, binds very tightly to
molybdenum-sulfide complex of the enzyme in active site, commits suicide
3. Heavy Metals – heavy-metal toxicity is cause by tight binding of a metal such as mercury, lead,
aluminum, or iron to a functional group in an enzyme
a. heavy metals are non-specific for enzymes they inhibit
i. mercury which binds to many enzymes at sulfhydryl groups in active site
ii. Lead inhibits through replacing normal functioning metal in enzyme (Ca, Fe, Zn)
1. Neural toxicity comes from replacing Ca2+ in CNS proteins
Basic Classes of Enzymes
1. Oxidoreductases – Oxidation/reduction reactions gain/lose electrons such as with NAD+/NADH
2. Transferases – transfer functional groups from one molecule to another (kinase if this is a
phosphate), (glycosyltransferase if this transfers a carbohydrate)
a. Transaminases – transfer of nitrogen
b. Synthase – when compound is synthesized
3. Hydrolase – catalyzes hydrolysis reactions C-O, C-N, and C-S bonds are cleaved by addition of
H2O in the form of OH- and H+
a. Protease – cleaves proteins such as chymotrypsin
4. Lyases – cleave C-C, C-O, and C-N by means other than hydrolysis or oxidation, such as
aldolases, decarboxylases, and thiolases
5. Isomerases – rearrange atoms in a molecule, mutases move phosphate from one to another
6. Ligases – synthesize C-C, C-S, C-O and C-N bonds in reactions coupled to cleavage of high-energy
phosphate from ATP, such as carboxylases