Concept 8.5: Regulation of enzyme activity helps control metabolism Chemical chaos would result if a cell’s metabolic pathways were not tightly regulated A cell does this by switching on or off the genes that encode specific enzymes or by regulating the activity of enzymes Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Allosteric Regulation of Enzymes Allosteric regulation may either inhibit or stimulate an enzyme’s activity Allosteric regulation occurs when a regulatory molecule binds to a protein at one site and affects the protein’s function at another site Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Allosteric Activation and Inhibition Most allosterically regulated enzymes are made from polypeptide subunits Each enzyme has active and inactive forms The binding of an activator stabilizes the active form of the enzyme The binding of an inhibitor stabilizes the inactive form of the enzyme Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Figure 8.20 Allosteric regulation of enzyme activity Allosteric enyzme with four subunits Active site (one of four) Regulatory site (one of four) Activator Active form Stabilized active form Oscillation Non- functional active site Inhibitor Inactive form Stabilized inactive form Figure 8.20 Allosteric regulation of enzyme activity (a) Allosteric activators and inhibitors Substrate Inactive form Stabilized active form (b) Cooperativity: another type of allosteric activation

Stabilized active form Fig. 8-20a Allosteric enzyme with four subunits Active site (one of four) Regulatory site (one of four) Activator Active form Stabilized active form Oscillation Figure 8.20a Allosteric regulation of enzyme activity Non- functional active site Inhibitor Inactive form Stabilized inactive form (a) Allosteric activators and inhibitors

Cooperativity is a form of allosteric regulation that can amplify enzyme activity In cooperativity, binding by a substrate to one active site stabilizes favorable conformational changes at all other subunits Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

(b) Cooperativity: another type of allosteric activation Fig. 8-20b Substrate Inactive form Stabilized active form Figure 8.20b Allosteric regulation of enzyme activity (b) Cooperativity: another type of allosteric activation

Identification of Allosteric Regulators Allosteric regulators are attractive drug candidates for enzyme regulation Inhibition of proteolytic enzymes called caspases may help management of inappropriate inflammatory responses Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Hypothesis: allosteric inhibitor locks enzyme in inactive form Fig. 8-21 EXPERIMENT Caspase 1 Active site Substrate SH SH Known active form Active form can bind substrate Allosteric binding site SH S–S Allosteric inhibitor Known inactive form Hypothesis: allosteric inhibitor locks enzyme in inactive form Figure 8.21 Are there allosteric inhibitors of caspase enzymes? RESULTS Caspase 1 Inhibitor Active form Allosterically inhibited form Inactive form

EXPERIMENT Allosteric binding site Allosteric inhibitor Caspase 1 Fig. 8-21a EXPERIMENT Caspase 1 Active site Substrate SH SH Known active form Active form can bind substrate Figure 8.21 Are there allosteric inhibitors of caspase enzymes? Allosteric binding site SH S–S Allosteric inhibitor Known inactive form Hypothesis: allosteric inhibitor locks enzyme in inactive form

RESULTS Caspase 1 Inhibitor Active form Allosterically inhibited form Fig. 8-21b RESULTS Caspase 1 Inhibitor Figure 8.21 Are there allosteric inhibitors of caspase enzymes? Active form Allosterically inhibited form Inactive form

Feedback Inhibition In feedback inhibition, the end product of a metabolic pathway shuts down the pathway Feedback inhibition prevents a cell from wasting chemical resources by synthesizing more product than is needed Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Fig. 8-22 Initial substrate (threonine) Active site available Threonine in active site Enzyme 1 (threonine deaminase) Isoleucine used up by cell Intermediate A Feedback inhibition Enzyme 2 Active site of enzyme 1 no longer binds threonine; pathway is switched off. Intermediate B Enzyme 3 Intermediate C Figure 8.22 Feedback inhibition in isoleucine synthesis Isoleucine binds to allosteric site Enzyme 4 Intermediate D Enzyme 5 End product (isoleucine)

Specific Localization of Enzymes Within the Cell Structures within the cell help bring order to metabolic pathways Some enzymes act as structural components of membranes In eukaryotic cells, some enzymes reside in specific organelles; for example, enzymes for cellular respiration are located in mitochondria Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Fig. 8-23 Mitochondria Figure 8.23 Organelles and structural order in metabolism 1 µm

Progress of the reaction Fig. 8-UN2 Course of reaction without enzyme EA without enzyme EA with enzyme is lower Reactants Free energy Course of reaction with enzyme ∆G is unaffected by enzyme Products Progress of the reaction

Fig. 8-UN3

Fig. 8-UN4

Fig. 8-UN5

You should now be able to: Distinguish between the following pairs of terms: catabolic and anabolic pathways; kinetic and potential energy; open and closed systems; exergonic and endergonic reactions In your own words, explain the second law of thermodynamics and explain why it is not violated by living organisms Explain in general terms how cells obtain the energy to do cellular work Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

Explain how ATP performs cellular work Explain why an investment of activation energy is necessary to initiate a spontaneous reaction Describe the mechanisms by which enzymes lower activation energy Describe how allosteric regulators may inhibit or stimulate the activity of an enzyme Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings