Catalysis: Control of Activity, Biochemistry

Catalysis: Control of Activity

1. Catalysis: Control of Activity by Dr. Asad Ullah Ph. D ( Food Science and Technology) M. Sc. ( Biochemistry )

2. Regulation of Enzyme Activity Apart from their ability to greatly speed the rates of chemical reactions in cells, enzymes have another property that makes them valuable. This property is that their activity which can be regulated, allowing them to be activated and inactivated, as necessary. This is tremendously important in maintaining homeostasis , permitting cells to respond in controlled ways to changes in both internal and external conditions. Inhibition of specific enzymes by drugs can also be medically useful. Understanding the mechanisms that control enzyme activity is, therefore , of considerable importance.

3. Enzyme Inhibition An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity Reversible and irreversible inhibitors bind to an enzyme to suppress its activity If inhibitor permanently bind to an enzyme. These types of inhibitors are called irreversible. If inhibitor transiently bind to an enzyme. These are called reversible. If inhibitor permanently binds to an enzyme… sucide inhibition. There are 4 types of inhibitor. Competitive (Reversible) Non-competitive (Reversible ) Uncompetitive (Reversible ) S uicide inhibition (Irreversible )

4. Competitive Inhibition Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an enzyme. Probably the easiest type of enzyme inhibition to understand is competitive inhibition and it is the one most commonly exploited pharmaceutically . An example is methotrexate, which resembles the folate substrate of the enzyme dihydrofolate reductase (DHFR ). This enzyme normally catalyzes the reduction of folate , an important reaction in the metabolism of nucleotides .

5. Km change but No effect on V max Reactions without inhibitor (with buffer and constant amounts of enzyme, varying amounts of substrate , equal reaction times) Red Line  Reactions performed in the same manner except that a fixed amount of the methotrexate inhibitor (blue line) ( remember that the inhibitor is at fixed concentration ).

6. In terms of competitive inhibitors, inhibitor compete directly with the substrate to bind to the active site of the enzyme . In turn, they will decrease the affinity of the enzyme for the substrate, which will increase the K M .

7. Non-competitive inhibition Noncompetitive inhibition , a type of allosteric regulation, is  a specific type of enzyme inhibition characterized by an inhibitor binding to an allosteric site resulting in decreased efficacy of the enzyme . An allosteric site is simply a site that differs from the active site- where the substrate binds.

8. With noncompetitive inhibition , increasing the amount of substrate has no effect on the percentage of enzyme that is active. Indeed, in noncompetitive inhibition , the percentage of enzyme inhibited remains the same through all ranges of [S]. V max is reduced in noncompetitive inhibition compared to uninhibited reactions.

9. Reducing the amoun t of enzyme present reduces V max Km for noncompetitively inhibited reactions does not change from that of uninhibited reactions. This is because, one can only measure the Km of active enzymes and Km is a constant for a given enzyme.

10. Example of Non-competitive Inhibitor Both alanine and ATP  act as non-competitive inhibitors of pyruvate kinase, the enzyme that catalyzes the final step in the glycolytic pathway

11. Uncompetitive inhibition A third type of enzymatic inhibition is that of uncompetitive inhibition , which has the odd property of a reduced V max as well as a reduced Km . The explanation for these seemingly odd results is rooted in the fact that the uncompetitive inhibitor binds only to the enzyme-substrate ( ES ) complex. ES-I complex cannot release product while the inhibitor is bound, thus explaining the reduced V max .

12. Suicide inhibition or   mechanism-based inhibition , is an irreversible form of enzyme inhibition that occurs when an enzyme binds a substrate analog and forms an irreversible complex with it through a covalent bond during the normal catalysis reaction. The inhibitor binds to the active site where it is modified by the enzyme to produce a reactive group that reacts irreversibly to form a stable inhibitor-enzyme complex. This usually uses a prosthetic group or a coenzyme, forming electrophilic alpha and beta unsaturated  carbonyl compounds and  imines. Some clinical examples of suicide inhibitors include: Disulfiram , which inhibits the acetaldehyde dehydrogenase enzyme. Aspirin , which inhibits  cyclooxygenase 1 and 2 enzymes . Penicillin, Penicillin, which inhibits  DD transpeptidase  from building bacterial  cell walls. Suicide inhibition or  Mechanism-based inhibition

13. In many enzymes, access of the substrate to the active site is limited by a barrier :  gatekeeper residues at the entrance to the binding pocket , a lid undergoing conformational transition between open and closed states, or domain rearrangements which control substrate access Substrate access: A third means of controlling enzymes Figure. Cellulose accessibility in enzymatic hydrolysis

14. Profiles of selected tunnel clusters T1-T3 were evaluated as to radius and distance from the active site during MD simulations of 2 μs length (see color scale ). Each line represents the tunnel profile of a single snapshot. Black dashed lines mark the average length of tunnels in their respective cluster. T1 is the longest and T3 the shortest of the three tunnels. Molecular dynamics simulations The substrate access to enzyme

15. REVERSIBLE INHIBITION The inhibitor binds reversibly to the enzyme at the same site as substrate. The inhibitor resemble S structurally. S-binding and I-binding are mutually exclusive, competitive processes. The inhibition is blocked when the substrate concentration increases. K mapp increases and V is unaffected Competitive Inhibition ENZYME KINETICS

16. Competitive Inhibition REVERSIBLE INHIBITION ENZYME KINETICS

17. Noncompetitive inhibition Inhibitor interacts with both E and ES. The inhibition is not blocked when the substrate concentration increases. V app decreases and K m is unaffected REVERSIBLE INHIBITION ENZYME KINETICS

18. Noncompetitive inhibition REVERSIBLE INHIBITION ENZYME KINETICS

19. Inhibitor only combines with ES It does not bind in the active site. V app and Km app decrease Uncompetitive inhibition REVERSIBLE INHIBITION ENZYME KINETICS

20. Uncompetitive inhibition REVERSIBLE INHIBITION ENZYME KINETICS

21. Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP) Ciclooxigenase inhibition by aspirin IRREVERSIBLE INHIBITION ENZYME KINETICS

22. Feedback Inhibition enzyme 2 enzyme 3 enzyme 4 enzyme 5 enzyme 1 SUBSTRATE END PRODUCT (tryptophan) A cellular change, caused by a specific activity, shuts down the activity that brought it about Feedback inhibition of enzyme activity occurs in pathways which require multiple enzyme-catalyzed steps The end product of the pathway inhibits the first enzyme in the pathway when there is an abundance of product the pathway is shut down

23. Regulation of Enzyme Action Some enzymes require cofactors to function normally. These are either metal ions or small organic molecules called coenzymes . The cofactors usually are in the active site and are involved in transition state stabilization. Enzyme activity can be regulated by molecules which are not part of the enzyme Competitive inhibition : molecule similar in size and shape to substrate binds to active site. “competes” with substrate for active site

24. Defined as the process by which the interaction of a chemical or protein ( ligand ) at one location on a protein or macromolecular complex ( the allosteric site ) influences the binding or function of the same or another chemical or protein at a topographically distinct site. Allosterism??

25. Allosteric Inhibition Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. In contrast, allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases

26. Allosteric Inhibitor Example ATP is an example of an allosteric inhibitor. The enzyme taking part in glycolysis is phosphofructokinase. It transforms ADP into ATP. When the concentration of ATP is too much in the system, then ATP functions as an allosteric inhibitor . ATP combines with phosphofructokinase and slows down the transformation of ADP. In this manner, ATP is inhibiting the unwanted generation of itself.

27. An interesting kind of allosteric control is exhibited by HMG-CoA reductase , which catalyzes an important reaction in the pathway leading to the synthesis of cholesterol. Binding of cholesterol to the enzyme reduces the enzyme’s activity significantly. Cholesterol is not a substrate for the enzyme, so it is therefore a heterotropic effector . Notably, though, cholesterol is the end product of the pathway that HMG-CoA reductase catalyzes a reaction in. When enzymes are inhibited by an end-product of the pathway in which they participate, they are said to exhibit feedback inhibition.

28. Another example of allosteric control and feedback inhibition is associated with Aspartate Transcarbamoylase ( ATCase ). The R-state of ATCase allows the substrate to have easier access to the six active sites and the reaction occurs more rapidly. For the same amount of substrate, an enzyme in the R-state will have a higher velocity than the same enzyme that is not in the R-state . By contrast, if the enzyme binds to CTP on one of its regulatory subunits, the subunits will arrange in the T-state and in this form, the substrate will not have easy access to the active sites, resulting in a slower velocity for the same concentration of substrate compared to the R-state. ATCase is interesting in that it can also flip into the R-state when one of the substrates (aspartate) binds to an active site within one of the catalytic subunits

29. Covalent Modifications A re   enzyme- catalysed alterations of synthesised proteins (enzyme) and include the addition or removal of chemical groups . Modifications can target a single or multiple AAs and will change the chemical properties of the site.

30. Phosphorylation/ dephosphorylation Another common mechanism for control of enzyme activity by covalent modification is phosphorylation. The phosphorylation of enzymes (on the side chains of serine, threonine or tyrosine residues) is carried out by protein kinases . Enzymes activated by phosphorylation can be regulated by the addition of phosphate groups by kinases or their removal by phosphatases . Thus, this type of covalent modification is readily reversible, in contrast to proteolytic cleavage. Ser , Thr , Tyr Ser , Thr , Tyr

31. Oxidation/Reduction An interesting covalent control of enzymes using oxidation/reduction is exhibited in photosynthetic plants. In the light phase of photosynthesis , electrons are excited by light and flow through carriers to NADP+, forming NADPH. When NADPH con. is high in the light reaction, the con. of reduced ferredoxin (a molecule donating electrons to NADP+) is also high. Reduced ferredoxin can transfer electrons to thioredoxin , reducing it. Reduced thioredoxin can, in turn, transfer electrons to proteins to reduce their disulfide bonds. Four enzymes related to the Calvin cycle can receive electrons from thioredoxin and become activated , as a result.

32. Enzyme Synthesis All enzymes are protein in nature, and their synthesis involves the linking together of AAs in correct sequence. Each organism has genome (DNA), coded information for the building up of its own specific proteins. The genome, or total stock of instructions for synthesis of metabolites, determines the patterns of proteins to be produced and in the final analysis determines the form of each species and the inherited constitution of the individual within the species . Enzymes are apparently synthesized not singly but as part of a sequence of the enzymes required for the successive steps in a metabolic pathway. A series of structural genes determines the molecular composition of the enzymes. From these genes, molecules of mRNA are transcribed And thus enzyme protein is synthesized with the assistance of tRNA and rRNA

33. Control of Enzyme Synthesis The rate of enzyme synthesis is under the control of regulator and operator genes, with a repressor molecule in the cell cytoplasm acting as a link between the two. There are two basic systems of control, the inducible system and the repressible system .

34. Control of Enzyme Synthesis Obviously, coordination and control of enzyme synthesis are essential for correct cellular function and at a given moment, most of the potentialities inherent in the genome must be inactive or repressed

35. Enzyme Induction In the inducible system, the repressor molecule is synthesised , under the coded instructions of a regulatory gene, and in its active form, it prevents the formation of specific proteins. To allow the formation of those proteins when they are required, the repressor is inactivated by combination with an inducer. As a result, an operator gene is allowed to switch on, setting in motion the transcription of a series of genes which code for single enzymes, or groups of related enzymes involved in a single metabolic pathway.

36. E nzyme Repression This is the usual process for catabolic enzyme sequences. As a theoretical example, one might consider tyrosine as the inducing substrate for a series of enzymes required to convert it to noradrenalin. Increasing concentration of tyrosine induces increased concentration of enzymes for its conversion. When all available tyrosine is converted, repressor molecules are free to attach to the operator gene once more, and inhibit enzyme synthesis. In the repressible enzyme system on the other hand, the repressor molecule is considered to be active only when combined with a corepressor, and it is the absence of the corepressor which initiates new enzyme synthesis, in a process known as derepression

37. Conclusion; Induction Vs Repression Properties Induction Repression Meaning and key elements The meaning of induction system states that it will induce the gene expression via an inducer. Key elements: Inducer or anti-repressor The meaning of repression system states that it will suppress the gene expression via a corepressor. Key elements: Corepressor or effector molecule Regulation The operon system regulates the synthesis of enzymes that are stimulated by the addition of inducer. Lactose operon (lac operon) The operon system suppresses the enzyme synthesis, which is facilitated by the existing end-product or corepressor molecules. Tryptophan operon (Tryp operon) Mechanism Inducer inactivates the repressor protein by preventing the attachment of a repressor to the operator region Corepressor activates the apo -repressor protein (inactive) and allows the attachment of an active repressor to the operator region Effect on the control system Mediates movement of the RNA polymerase along the control system, i.e. promoter and operator region Blocks the movement of RNA polymerase along the promoter and operator region Effect on structural genes It initiates the expression of structural genes It inhibits the expression of structural genes Overall impact An induction system activates or turns on the whole operon system through an adequate supply of the inducer metabolites A repression system terminates or switches off the entire operon system under an adequate level of corepressor molecules Operon system The genetic system regulated by the presence or absence of an inducer is called inducible operon The genetic system regulated by the presence or absence of a corepressor is called repressible operon Enzyme Enzymes synthesis stimulated by the addition of inducer metabolites are termed as inducible enzymes Enzymes synthesis inhibited by the addition of corepressor molecules are termed as repressible enzymes Metabolic pathway It operates a catabolic synthesis It operates an anabolic synthesis