Lecture 16:Regulation of Proteins 3:

Isozymes and Covalent Modification

Isozymes

Covalent Modification

Protein Kinase A

Biological Processes are Carefully Regulated

Allosteric Control: The activity of some proteins can be controlled by modulatingthe levels of small signalling molecules. The binding of thesemolecules causes conformational changes in the proteinwhich affect its activity.

Multiple forms of Enzymes:Different tissues or developmental stages sometimes have specificversions of a given enzyme which have distinct properties althoughthey may have the same basic activity.

Reversible Covalent Modification:The activity of many proteins is controlled by attachment of smallchemical groups. The most common such modification isphosphorylation- attachment of a phosphate group.

Proteolytic Activation:Some enzymes are synthesized in an inactive form and must beactivated by cleavage of the inactive form.

Multiple Forms of Proteins

Different tissues or developmental stages differ in their requirementsfor the activity of various proteins.

An example is provided by fetal hemoglobin. Oxygen is provided tothe fetus through the mother’s circulatory system, which requiresthat oxygen be transferred from the maternal hemoglobin to the hemoglobin in the bloodstream of the fetus. The fetus expressesa different form of hemoglobin which has a higher oxygen affinitythan adult hemoglobin, facilitating this transfer.

This regulatory strategy is also a common means of controllingthe activity of enzymes.

Enzymes that carry out the same chemical reaction, but differ insequence, kinetic properties, and/or in regulatory properties,are called isozymes.

Isozymes of Lactate Dehydrogenase

Lactate dehydrogenase (LDH) functions in glucose metabolism.

Mammals have 2 versions of LDH, the H isozyme (found in heart)and the M isozyme (found in skeletal muscle) which are closelyrelated, sharing about 75% sequence identity.

LDH functions as a tetramer, but individual tetramers can becomposed of any combination of M and H isozymes. (M4, M3H, M2H2, MH3, or H4)

The M4 tetramer functions optimally under anaerobic conditions, while the H4 tetramer functions optimally underaerobic conditions.

The H4 tetramer has higher substrate affinity and also can beallosterically inhibited by pyruvate. The M4 tetramer has a loweraffinity for substrate and is not allosterically regulated.

The mixed tetramers exhibit properties intermediate between thethe H4 and M4 tetramers- differential expression of the two isozymesallows fine control over the LDH activity.

Developmental Regulation of LDH Isozymes

Age (days)

H4

M4

At different developmental stages in the rat heart, the two isozymes ofLDH are expressed to different degrees. In the early embryo the Mform predominates, and in the adult the H form predominates.

(anaerobic) (aerobic)

Tissue-Specific LDH Isozyme Expression

The levels of the different isozymes also vary from tissue to tissue.There is a distribution of different complexes in a given tissue.

LDH-1

LDH-2

LDH-3

LDH-4

LDH-5

Disruption of cells can lead to release of LDH into the bloodstream.In blood, the level of LDH-2 is normally higher than the level of LDH-1.But during a heart attack, LDH-1 can be released into the bloodstream,increasing it above the level of LDH-2. Analysis of the relative levelsof LDH isozymes serves as a diagnostic for a variety of medical conditions.

Covalent Modification

The activity of many proteins is controlled by various forms of covalentmodification- attachment of a small functional group. The modificationcan increase or suppress the activity of the proteins.

Specific enzymes carry out these modifications, and other enzymesremove them.

In most cases the modification is reversible- removal of the attachedgroup reverses the effect of the modification.

In this way the regulated proteins can be “turned on” and “turned off”as appropriate, depending on the cell’s need for that particular activity.

Examples of Covalent Modification

A variety of modifications exist, acting to control a wide rangeof cellular functions.

Acetylation of Histones

Histones are important eukaryotic proteins involved in the packagingof DNA in chromosomes and also in regulation of gene expression.

Histones are basic proteins, rich in Lys and Arg residues. The positivecharge assists in binding DNA, which is negatively charged.

The lysine residues of histones near regions of actively transcribed genesare heavily acetylated.

Histones DNA Section ofchromatin fiber

Acetylation of lysines in histones removes the positive charge, weakeningthe affinity of histones for DNA. This makes it easier to remove or displacethe histone from DNA to enable genes to be transcribed.

The covalent modfication of histones is carried out by enzymes calledhistone acetyltransferases. Removal of the acetyl group is carried outby deacetylases. These enzymes allow changes in the expressionlevel of genes in various locations on the chromosome.

In turn, the acetyltransferases and deacetylases are themselves regulatedby phosphorylation.

Phosphorylation

Probably the most widespread method of regulation isphosphorylation- attachment of a phosphate group.

A wide variety of enzymes, many membrane channels and transcriptionfactors, and virtually all metabolic processes are regulatedby phosphorylation.

Phosphorylation is carried out by protein kinases, enzymes whichtransfer a phosphate group from ATP to hydroxyl groups in proteins.

Kinases

A kinase is any enzyme which transfers a phosphate group. Kinasesare named for the target which receives the phosphate. (eg glycerolkinase phosphorylates glycerol)

The phosphate group derives from ATP, which is only present insidecells. Extracellular proteins are not regulated by phosphorylation.

An enzyme that phosphorylates a protein is a protein kinase.

Two major classes of protein kinases:

Serine/threonine kinases Group that receives the phosphate is a serine or threonine hydroxyl.

Tyrosine kinases.Group that receives the phosphate is a tyrosine hydroxyl.

Examples of Protein Kinases

Protein kinases comprise one of the largest protein families known- thereare more than 500 in humans. These allow regulation specific to particulartissues, developmental stages, and substrate proteins.

Kinases themselves are controlled by many different kinds of cellularsignals including by other kinases.

Kinases and Phosphatases

Phosphorylation is carried out by kinases. Removal of phosphate groups is carried out by phosphatases.These reactions are not the reverse of one another.Both reactions are energetically downhill and so are unidirectionalbut proceed extremely slowly in the absence of enzymes.

Kinase

Phosphatase

Transfer of phosphatefrom ATPto protein.

Transfer of phosphatefrom proteinto water.

Effects of Phosphorylation

Phosphorylation often activates a target molecule, for example byinducing a conformational change to a more active state. The influenceon target activity can be accomplished through various effects.

Charge: Phosphorylation adds 2 negative charges which canparticipate in (or disrupt) charge-charge interactions.

Hydrogen bonding: The phosphate group can participate in 3 or morehydrogen bonds.

Energy: The substantial amount of energy in the phosphate bondcan strongly affect conformational equilibria.

Time: Phosphorylation can be accomplished in seconds and can last foras long as needed. By regulating the phosphorylation and dephosphorylationsteps, the activity of the target can be adjusted to synchronize with aphysiological process.

Amplification: A single kinase can phosphorylate and activate hundreds oftarget molecules resulting in a large effect from a small stimulus.

Specificity of Protein Kinases

Dedicated protein kinases phosphorylate only a single target or a fewclosely related ones, allowing fine control over this limited target.

Multifunctional protein kinases phosphorylate many different targets,allowing a single kinase to control a variety of different processes.

For kinases with many different targets, comparison of the amino acidsequences of the residues near the phosphorylation sites identifiespatterns. The primary determinant of specificity is the amino acidssurrounding the phosphorylation site.

For example, protein kinase A recognizes and phosphorylates the serine or threonine in the sequences:

Arg-Arg-X-Ser-Z or Arg-Arg-X-Thr-Z

where X is a small residue (eg Gly)and Z is a large hydrophobic residue (eg Ile)

Protein Kinase A

Many physiological processes are regulated by hormones, which areare extracellular signalling molecules.

Hormones bind to receptors at the cell surface. In some cases, thebinding of hormones causes the formation of intracellular signallingmolecules, or second messengers, such as cyclic AMP.

This carries the signal from the hormone into the cell and results inactivation of many different proteins. Most of these proteins areactivated by a single kinase, protein kinase A. (PKA)

Protein Kinase A is Regulated by cAMP

PKA consists of:2 C subunits (catalytic subunits) that contain the kinase activity2 R subunits (regulatory subunits) that bind cAMP

In the absence of cAMP, an inactive C2R2 complex is formed in which theregulatory subunits tightly bind and sequester the catalytic subunits.

In the presence of cAMP (eg in response to a hormone) the regulatorysubunit binds the cAMP, inducing a conformational change that releasesthe catalytic subunits, which can then begin phosphorylating their targets.

Mechanism of cAMP Regulation

The R subunit of PKA contains a sequence nearly matching the preferredsubstrate of the C subunits but incapable of being phosphorylated.

This pseudo-substrate sequence contains the residues …Arg-Arg-Gly-Ala-Ile…

and the C subunit binds this sequence tightly at its active site, preventingother substrates from being phosphorylated.

The binding of cAMP by the R subunit causes an allosteric conformational shift which removes the pseudo-substrate sequence fromthe C subunit, releasing it.

Structural Basis of cAMP Regulation

The structure of the catalytic subunit of PKA in complex with apseudosubstrate peptide allows the substrate specificity and inhibitionby the R subunit to be understood as due to complementarity withspecific residues in the c subunit.

Hydrophobic interactions

Ionicinteractions

Arg-Arg-Asn-Ala-Ile

Catalytic subunit

Summary:

Isozymes are enzymes which have the same activity but different kineticsor regulatory properties- differential expression of isozymes allows controlover enzyme activity.

Many proteins are regulated by covalent modification. The most commonsuch modification is phosphorylation.

Protein kinase A carries out phosphorylation of a wide variety of targets in response to cyclic AMP.

Key Concepts:Isozymes of LDHRegulation of histones by acetylationKinasesPhosphatasesRole of Protein Kinase A