Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter SevenThe Behavior of Proteins:

Enzymes, Mechanisms, and Control

Allosteric Enzymes

• Allosteric:Allosteric: Greek allo + steric, other shape• Allosteric enzymeAllosteric enzyme:: an oligomer whose biological activity is affected by

other substances binding to it

• these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure

• Allosteric effectorAllosteric effector:: a substance that modifies the behavior of an allosteric enzyme; may be an

• allosteric inhibitor

• allosteric activator• Aspartate transcarbamoylase (ATCase)Aspartate transcarbamoylase (ATCase)

• feedback inhibitionfeedback inhibition

Feedback Inhibition

Formation of product inhibits its continued production

ATCase

• Rate of ATCase catalysis vs substrate concentration

• Sigmoidal shape of curve describes allosteric behavior

• ATCase catalysis in presence of CTP; ATP

ATCase (Cont’d)

• Organization of ATCase• catalytic unit: 6 subunits

organized into 2 trimers• regulatory unit: 6 subunits

organized into 3 dimers

• Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cysteine (p-hydroxymercuribenzoate)

Allosteric Enzymes (Cont’d)

• Two types of allosteric enzyme systems exist

Note: for an allosteric enzyme, the substrate concentration at one-half Vmax is called the K0.5

• K system:K system: an enzyme for which an inhibitor or activators alters K0.5

• V system:V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5

Allosteric Enzymes (Cont’d)

• The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme

• allosteric effector:allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme

• homotropic effects:homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase

• heterotropic effects:heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP

Summary: Homo and Hetero modulators

Homotropic•A homotropic allosteric modulator is a substratesubstrate for its target enzyme, as well as a regulatory molecule of the enzyme's activity. It is typically an activator of the enzyme. For example, O2 is a homotropic allosteric modulator of hemoglobin.

Heterotropic•A heterotropic allosteric modulator is a regulatory molecule that is not also the enzyme's substrate. It may be either an activator or an inhibitor of the enzyme. For example, H+, CO2, and 2,3-bisphosphoglycerate are heterotropic allosteric modulators of hemoglobin.[6]

•Some allosteric proteins can be regulated by both their substrates and other molecules. Such proteins are capable of both homotropic and heterotropic interactions.

The Concerted Model

• Wyman, Monod, and Changeux - 1965

• The enzyme has two conformations

• R (relaxed):R (relaxed): binds substrate tightly; the active form

• T (tight or taut):T (tight or taut): binds substrate less tightly; the inactive form

• in the absence of substrate, most enzyme molecules are in the T (inactive) form

• the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form

• in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted

Concerted Model (Cont’d)• A model represented by a protein having two conformations• Active (R) form-Relaxed binds substrate tightly, Inactive (T) form- Tight (taut) binds substrate less tightly both change from T to R at the same time• Also called the concerted model• Substrate binding shifts equilib. To the relaxed state.

Any unbound R is removed KR<KT

Ratio of dissociation constants is called c

The Monod-Wyman-Changeaux modelWhen substrate binds R, it removesfree R. This makes more R (from T) to re-establish equilib.= allosteric effect

C= KR/KL L =T/R

Concerted Model (Cont’d)

• The model explains the sigmoidal effects

• Higher L means higher favorability of free T form

• Higher c means higher affinity between S and R form, more sigmoidal as well.

C= KR/KL L =T/R

Concerted Model (Cont’d)

• An allosteric activator (A) binds to and stabilizes the R (active) form• An allosteric inhibitor (I) binds to and stabilizes the T (inactive) form

Effect of binding activators and inhibitors

Sequential Model (Cont’d)

• Main Feature of Model:

• the binding of substrate induces a conformational change from the T form to the R form

• the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding

• sequential model represents cooperativity

Sequential Model (Cont’d)Sequential model for cooperative binding of substrate to an allosteric enzyme

• R form is favored by allosteric activator

• Allosteric inhibition also occurs by the induced-fit mechanism

• Unique feature of Sequential Model of behavior: Negative cooperativity- Induced conformational changes that make the enzyme less likely to bind more molecules of the same type.

• Sequential Model:When S Binds, Affinityincreases as more subunits assume R state

When I Binds, Affinity for Sdecreases as more subunits assume T state

Summary: Concerted versus Sequential

Concerted model•also referred to as the symmetry model postulates that enzyme subunits are connected in such a way that a conformational change in one subunit is necessarily conferred to all other subunits. Thus, all subunits must exist in the same conformation. In the absence of any ligand (substrate or otherwise), the equilibrium favours one of the conformational states, T or R. The equilibrium can be shifted to the R or T state through the binding of one ligand (the allosteric effector or ligand) to a site that is different from the active site (the allosteric site).

Sequential model•Subunit are not connected in such a way that a conformational change in one induces a similar change in the others. Thus, all enzyme subunits do not necessitate the same conformation. Moreover, the sequential model dictates that molecules of substrate bind via an induced fit protocol. In general, when a subunit randomly collides with a molecule of substrate, the active site, in essence, forms a glove around its substrate. While such an induced fit converts a subunit from the tensed state to relaxed state, it does not propagate the conformational change to adjacent subunits. Instead, substrate-binding at one subunit only slightly alters the structure of other subunits so that their binding sites are more receptive to substrate. To summarize: 1) subunits need not exist in the same conformation2) molecules of substrate bind via induced-fit protocol 3) conformational changes are not propagated to all subunits 4) substrate-binding causes increased substrate affinity in adjacent subunits.

Control of Enzyme Activity via Phosphorylation

• The side chain -OH groups of Ser, Thr, and Tyr can form phosphate esters

• Phosphorylation by ATP can convert an inactive precursor into an active enzyme

• Membrane transport is a common example

Membrane Transport

Source of PO4 is ATP

• When ATP is hydrolyzed, energy released that allows other energetically unfavorable reactions to take place

• PO4 is donated to residue in protein by protein kinases

Zymogens

• Zymogen:Zymogen: Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme

• Chymotrypsinogen

• synthesized and stored in the pancreas

• a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds

• when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin

Activation of chymotrypsin

• Activation of chymotrypsinogen by proteolysis

Chymotrypsin

• A15-unit polypeptide remains bound to -chymotrypsin by a single disulfide bond

-chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give -chymotrypsin

-chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds

• changes in 1°structure that accompany the change from chymotrypsinogen to -chymotrypsin result in changes in 2°- and 3°structure as well.

-chymotrypsin is enzymatically active because of its 2°- and 3°structure, just as chymotrypsinogen was inactive because of its 2°- and 3°structure

The Active Site

Some important questions to ask about enzyme mode of action:• Which amino acid residues on an enzyme are in the active site

and catalyze the reaction?

• What is the spatial relationship of the essential amino acids residues in the active site?

• What is the mechanism by which the essential amino acid residues catalyze the reaction?

• As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Phe or Tyr

Kinetics of Chymotrypsin Reaction

• p-nitrophenyl acetate is hydrolyzed by chymotrypsin in 2 stages.

• At the end of stage 1, the p-nitrophenolate ion is released.

• At stage 2, acyl-enzyme intermediate is hydrolyzed and acetate (Product) is released…free enzyme is regenerated

Chymotrypsin

• Reaction with a model substrate

Chymotrypsin (Cont’d)

• Chymotrypsin is a serine protease

• DIPF inactivates chymotrypsin by reacting with serine-195, verifying that this residue is at the active site

Chymotrypsin (Cont’d)

• H57 also critical for activation of enzyme

• Can be chemically labeled by TPCK

Chymotrypsin (Cont’d)

• Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site

• Results of x-ray crystallography show the definite arrangement of amino acids at the active site

• In addition to His-57 and Ser-195, Asp-102 is also involved in catalysis at the active site

• The folding of the chymotrypsin backbone, mostly in antiparallel pleated sheet array, positions the essential amino acids around the active-site pocket

Chymotrypsin (Cont’d)

• The active site of chymotrypsin shows proximity of 2 reactive a.a.

Mechanism of Action of Critical Amino Acids in Chymotrypsin

• Serine oxygen is nucleophile

• Attacks carbonyl group of peptide bond

Catalytic Mechanisms

General acid-base catalysis:General acid-base catalysis: depends on donation and acceptance of protons (proton transfer reactions)

• Nucleophilic substitution catalysts- Nucleophilic electron-rich atom attacks electron deficient atom.

• same type of chemistry can occur at active site of

enzyme: SN1, SN2

Catalytic Mechanisms (Cont’d)

• Lewis acid/base reactions

• Lewis acid:Lewis acid: an electron pair acceptor

• Lewis base:Lewis base: an electron pair donor

• Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes (metal ion catalysts)

• carboxypeptidase A requires Zn2+ for activity

Catalytic Mechanisms (Cont’d)

• Zn2+ of carboxypeptidase is complexed with:

• The imidazole side chains of His-69 and His-196 and the carboxylate side chain of Glu-72

• Activates the carbonyl group for nucleophilic acyl substitution

Enzyme Specificity

• Absolute specificityAbsolute specificity: catalyzes the reaction of one unique substrate to a particular product

• Relative specificityRelative specificity:: catalyzes the reaction of structurally related substrates to give structurally related products

• Stereospecificity:Stereospecificity: catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed

• example: hydration of a cis alkene (but not its trans isomer) to give an R alcohol (but not the S alcohol)

Asymmetric binding

• Enzymes can be stereospecific (Specificity where optical activity may pay a role)

• Binding sites on enzymes must be asymmetric

Active Sites and Transition States

• Enzyme catalysis

• an enzyme provides an alternative pathway with a lower activation energy

• the transition state often has a different shape than either the substrate(s) or the product(s)

• “True nature” of transition state is a chemical species that is intermediate in structure between the substrate and the product.

• Transition state analog:Transition state analog: a substance whose shape mimics that of a transition state

• In 1969 Jenks proposed that

• an immunogen would elicit an antibody with catalytic activity if the immunogen mimicked the transition state of the reaction

• the first catalytic antibody or abzyme was created in 1986 by Lerner and Schultz

*(Biochemical Connections, p. 196)

Coenzymes

• Coenzyme:Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction• metal ions- can behave as coordination compounds. (Zn2+,

Fe2+)• organic compounds, many of which are vitamins or are

metabolically related to vitamins (Table 7.1).

NAD+/NADH

• Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology.

• Contains:

1) nicotinamide ring

2) Adenine ring

3) 2 sugar-phosphate groups

NAD+/NADH (Cont’d)

• NAD+ is a two-electron oxidizing agent, and is reduced to NADH

• Nicotinamide ring is where reduction-oxidation occurs

B6 Vitamins

• The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another.

• Important in amino acid biosynthesis

Pyridoxal Phosphate

• Pyridoxal and pyridoxamine phosphates are involved in the transfer of amino groups in a reaction called transaminationtransamination

Figure 7.21 p. 197

Ribozymes

• The first ribozymes discovered included those that catalyze their own self-splicing

• More recently, ribozymes have been discovered that are involved in protein synthesis

• Group I ribozymes • require an external guanosine

• example: pre-rRNA of the protozoan Tetrahymena (next screen)

• Group II ribozymes• display a lariat mechanism similar to mRNA splicing

• no requirement for an external nucleotide

Self-splicing of pre-rRNA (Group I)

Also termed catalytic RNA, ribozymes function within the ribosome (as part of the large subunit ribosomal RNA) to link amino acids during protein synthesis, (also in a variety of RNA processing reactions: - RNA splicing, - viral replication, - tRNA biosynthesis.