Chapter 3

PROTEIN FUNCTION

All proteins bind to other molecules

Binding is selective

The protein’s conformation determines its chemistry

cAMP-binding site in a protein

Chemistry at an active siteA “catalytic triad” in serine proteases. The side chain of Asp 102 induces His 57to remove the proton from Ser 195. The activated serine in turn forms a covalent

bond with the enzyme substrate, hydrolyzing a peptide bond

Sequence comparisons between protein family members highlight crucialligand binding sites

“Evolutionary tracing” identifies sites in a protein domain that are most crucial to the domain’s function

Proteins bind to other proteins through several types of interfaces

Surface-string interaction allows the SH2 domain to recognizea phosphorylated polypeptide as a loop on a second protein

Binding sites in proteins – each antibody recognizes a specific target

Noncovalent bonds mediate interactions between macromolecules

Binding strength is measured by the Equilibrium constant

Greater the equilibrium constant, greater the binding strength. This is a direct measure of the free-energy difference between the bound andthe free states

Enzymes are powerful and highly specific catalysts

Km is a measure of how tightly the substrate is bound.A large value of Km corresponds to weak binding

Substrate binding is the first step in enzyme catalysis

Enzymes speed reactions by selectively stabilizing transition states or in other words by decreasing the activation energy

The free energy required to attain the most unstable transition state is called theactivation energy for the reaction, and it is the major determinant of the reaction rate

One of the most important factors in enzyme catalysis is the ability of an enzyme to bind substrate more tightly in its transition state than in its ground state.

The difference in binding energy between these states lowers the activation energy of the reaction. This is illustrated by energy profiles for an enzyme in its wild-type form (a), for a mutant that stabilizes the substrate

in its transition state and therefore decreases the activation energy from ES to the transition state EST giving higher rates (b), and for a mutant that stabilizes the substrate in its ground state giving lower rates (c)

The stabilization of a transition state by an antibody creates an enzyme – creation of catalytic antibodies

The spontaneous reaction rates and the corresponding enzyme-catalyzed rates

Rate accelerations of 109 – 1023 are observed with enzymes compared to a 104

increase observed with catalytic antibodies

Enzymes can use simultaneous acid and base catalysis

Serine proteinases catalyze the hydrolysis of peptide bonds within a polypeptide chain.

The bond that is cleaved is called the scissile bond. (RA)x and (RB)y represent polypeptide chains of varying lengths

Schematic diagrams of the specificity pockets of chymotrypsin, trypsin and elastase, illustrating the preference for a side chain adjacent to the scissile bond in polypeptide

substrates.

Chymotrypsin prefers aromatic side chains and trypsin prefers positively charged side chains that can interact with Asp 189 at the bottom of the specificity pocket. The pocket is blocked

in elastase, which therefore prefers small uncharged side chains

Schematic diagram of the structure of chymotrypsin, which is folded into two antiparallel domains.

The six strands of each domain are red, the side chains of the catalytic triad are dark blue, and the disulfide bridges that join the three polypeptide chains are marked in violet. Chain A

(green, residues 1-13) is linked to chain B (blue, residues 16-146) by a disulfide bridge between Cys 1 and Cys 122. Chain B is in turn linked to chain C (yellow, residues 149-245) by a

disulfide bridge between Cys 136 and Cys 201. Dotted lines indicate residues 14-15 and 147-148 in the inactive precursor, chmotrypsinogen. These residues are excised during the

conversion of chymotrypsinogen to the active enzyme chymotrypsin

Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3- 4 and 5-6, red) of the two

domains.

These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket

A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step.

The four essential features of the serine proteinases are highlighted in yellow: the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-

chain substrate binding

(a) Formation of an acyl-enzyme intermediate involving a reactive Ser residue of the enzyme is the first step in hydrolysis of peptide bonds by serine proteinases.

First, a transition state is formed where the peptide bond is cleaved in which the C1 carbon has a tetrahedral geometry with bonds to four groups, including the reactive Ser residue of the

enzyme and a negatively charged oxygen atom. (b) Deacylation of the acyl-enzyme intermediate is the second step in hydrolysis. This is essentially the reverse of the acylation

step, with water in the role of the NH2 group of the polypeptide substrate. The base shown in the figure is a His residue of the protein that can accept a proton during the formation of the

tetrahedral transition state

The reaction catalyzed by lysozyme

Single Protein crystal of LysozymePhotographed by Mathias Klode

From Wikipedia, the free encyclopedia

Lysozyme

Schematic diagram of the structure of lysozyme which folds into two domains.

One domain is essentially -helical whereas the second domain comprises a three stranded antiparallel sheet and two helices. There are three disulfide bonds

(green), two in the -helical domain and one in the second domain.

Nature, 23 August 2001, Vol 412, 835-838

Some general strategies of enzyme catalysis

Multienzyme complexes help to increase the rate of cell metabolism

The structure of pyruvate dehydrogenase

Catalytic activities of enzymes are regulated

Feedback inhibition

Multiple feedback inhibition

Allosteric enzymes have two or more binding sites thatinteract with each other

Positive regulation caused by conformational coupling at two distant binding sites

Negative regulation caused by conformational coupling at two distant binding sites

Symmetric protein assemblies produce cooperative allosteric transitions

A cooperative allosteric transition in an enzyme composed of two identical subunits

The allosteric transitionin aspartate transcarbamoylaseis understood in atomic detail

Carbamoylphosphate +

aspartate

N-carbamoylaspartate

Many changes in proteins are driven by phosphorylation

Three-dimensional structure of a protein kinase

Cdk protein as an integrating device

Structure of Ras in its GTP-bound form

A comparison of the two major intracellular signaling mechanismsIn eucaryotic cells

An aminoacyl tRNA molecule bound to EF-Tu

The three-dimensional structure of EF-Tu with GTP bound

Change in the conformation of the switch helix in domain 1 causes domain 2and 3 to rotate as a single unit leading to the release of the tRNA

An allosteric “walking” protein – random walk

An allosteric motor protein

Transport of calcium ions by the Ca2+ pump

A protein interaction map ofDrosophila melanogaster