PROTEIN PHYSICSPROTEIN PHYSICS

LECTURE 24LECTURE 24LECTURE 24LECTURE 24

PROTEINS AT ACTION:PROTEINS AT ACTION:PROTEINS AT ACTION:PROTEINS AT ACTION:

BINDBIND TRANSFORMTRANSFORM RELEASERELEASEBIND BIND TRANSFORM TRANSFORM RELEASE RELEASE

BIND: repressorsBIND: repressors

αα-- turnturn--αα

ZnZn--fingersfingersfingersfingers

DNA & RNADNA & RNABINDINGBINDINGBINDINGBINDING

LeuLeu--zipperzipperLeuLeu zipperzipper

BINDBIND TRANSFORM TRANSFORM BINDBIND:: RepressorsRepressors

--BINDINGBINDING--INDUCED DEFORMATION INDUCED DEFORMATION MAKES REPRESSOR ACTIVE, and IT BINDS TO DNAMAKES REPRESSOR ACTIVE, and IT BINDS TO DNA

BIND:BIND: ImmunoglobulinsImmunoglobulins

ImmunoglobulinImmunoglobulin

BIND BIND TRANSFORM TRANSFORM RELEASERELEASE::ENZYMESENZYMESENZYMESENZYMES

chymotrypsinchymotrypsin

Note small Note small active siteactive site

Chymotrypsin catalyses hydrolysis of a peptide Chymotrypsin catalyses hydrolysis of a peptide

Spontaneous hydrolysis: very slowSpontaneous hydrolysis: very slow

Ch t iCh t iChymotrypsinChymotrypsinChymotrypsin is one of the serine proteases. Chymotrypsin is one of the serine proteases. Chymotrypsin is selective for peptide bonds withChymotrypsin is selective for peptide bonds withChymotrypsin is selective for peptide bonds with Chymotrypsin is selective for peptide bonds with aromatic or large hydrophobic side chains, such as aromatic or large hydrophobic side chains, such as Tyr, Trp, Phe and Met, which are on the carboxyl side Tyr, Trp, Phe and Met, which are on the carboxyl side of this bond. It can also catalyze the hydrolysis of of this bond. It can also catalyze the hydrolysis of easter bond. easter bond. The main catalytic driving force for Chymotrypsin is The main catalytic driving force for Chymotrypsin is the set of three amino acid known as catalytic triad. the set of three amino acid known as catalytic triad. This catalytic pocket is found in the whole serineThis catalytic pocket is found in the whole serineThis catalytic pocket is found in the whole serine This catalytic pocket is found in the whole serine protease family. protease family.

Properties of an Active SiteProperties of an Active SiteProperties of an Active SiteProperties of an Active Site

A shape that fits a specific substrate or A shape that fits a specific substrate or substrates onlysubstrates onlyyySide chains that attract the enzyme Side chains that attract the enzyme particular substrateparticular substrateparticular substrateparticular substrateSide chains specifically positioned to Side chains specifically positioned to speed the reactionspeed the reaction

The Catalytic TriadThe Catalytic TriadThe Catalytic TriadThe Catalytic Triad

chymotrypsinchymotrypsin

CHAIN CUTCHAIN CUT--INDUCED DEFORMATIONINDUCED DEFORMATIONMAKES ENZYME ACTIVEMAKES ENZYME ACTIVEMAKES ENZYME ACTIVEMAKES ENZYME ACTIVE

nonnon--activeactive↑↑

titi active active cat. sitecat. siteactive active

cat. sitecat. site

ChymotripsinChymotripsin ChymotripsinoChymotripsinogengeny py p y py p gg

SERSER--protease: protease: catalysiscatalysis

Chymotrypsin Protein Chymotrypsin Protein y ypy ypHydrolysisHydrolysis

Stage #1g

Chymotrypsin Protein Chymotrypsin Protein HydrolysisHydrolysis

Stage #2g

Chymotrypsin Protein Chymotrypsin Protein HydrolysisHydrolysis

Stage #3g

Chymotrypsin Protein Chymotrypsin Protein HydrolysisHydrolysis

Stage #4g

Chymotrypsin Protein Chymotrypsin Protein HydrolysisHydrolysis

Stage #5g

Chymotrypsin Protein Chymotrypsin Protein HydrolysisHydrolysis

Stage #6g

Transition State Transition State Stabilization Stabilization

Chymotrypsin KineticsChymotrypsin KineticsC y ot yps et csC y ot yps et cs

The initial "burst" inThe initial burst in chymotrypsin-catalysed hydrolysis of the p-nitrophenyl acetate

CHYMOTRYPSIN ACTIVE SITECHYMOTRYPSIN ACTIVE SITE with with INHIBITOR INHIBITOR

Catalytic antibodiesCatalytic antibodiesABZYM = AABZYM = AntyntyBBodyody enenZYMZYMABZYM AABZYM AntyntyBBodyody enenZYMZYM

Transition state (TS)Transition state (TS)Transition state (TS)Transition state (TS)

PreferentialPreferentialbinding binding

AntibodiesAntibodies

ggof TS:of TS:RIGIDRIGIDAntibodiesAntibodies

are are selectedselected

RIGIDRIGIDenzymeenzymeselectedselected

to TSto TS--likelikemoleculemolecule