Semarang University State Enzyme Catalysis Prof. Dr. Supartono, M.S Postgraduate Semarang University State

To understand how enzymes work at the molecular level. Objective To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn much through biochemical analysis.

To Be Explained Specificity Catalysis Regulation For specific substrates Amino acids residues involved Catalysis Amino acids involved Specific role(s) Regulation 27/11/2018 Supartono

Enzymes Enzymes are catalysts. They increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. Enzymes are neither used up in the reaction, nor do they appear as reaction products. 27/11/2018 Supartono

Discovery of Enzymes 1825 Jon Jakob Berzelius discovered the catalytic effect of enzymes. 1926 James Sumner isolated the first enzyme in pure form. 1947 Northrup and Stanley together with Sumner were awarded the Nobel prize for the isolation of the enzyme pepsin. Berzelius Sumner Northrup 27/11/2018 Stanley Supartono

Enzyme Characteristics High molecular weight proteins with masses ranging from 10,000 to as much as 2,000,000 grams per mole Substrate specific catalysts Highly efficien, increasing reaction rates by a factor as high as 108 27/11/2018 Supartono

Enzyme Nomenclature The earliest enzymes that were discovered have common names: i.e. Pepsin, Renin, Trypsin, Pancreatin The enzyme name for most other enzymes ends in “ase” The enzyme name indicates the substrate acted upon and the type of reaction that it catalyzes 27/11/2018 Supartono

Enzyme Names Examples of Enzyme Names Glutamic Oxaloacetic Transaminase (GOT) L-aspartate: 2-oxoglutarate aminotransferase. Enzyme names tend to be long and complicated. They are often abbreviated with acronyms 27/11/2018 Supartono

Enzyme Mechanisms Enzymes lower the activation energy for reactions and shorten the path from reactants to products 27/11/2018 Supartono

Enzyme Mechanisms E + S  ES  E + P The basic enzyme reaction can be represented as follows: E + S  ES  E + P Enzyme Substrate Enzyme substrate Enzyme Product(s) complex The enzyme binds with the substrate to form the Enzyme-Substrate Complex. Then the substrate is released as the product(s). 27/11/2018 Supartono

Enzyme Mechanisms E+SES E+P Diagram of the action of the enzyme sucrase on sucrose. E+SES E+P 27/11/2018 Supartono

Enzyme Specificity The action of an enzyme depends primarily on the tertiary and quaternary structure of the protein that constitutes the enzyme. The part of the enzyme structure that acts on the substrate is called the active site. The active site is a groove or pocket in the enzyme structure where the substrate can bind. 27/11/2018 Supartono

Cofactors Cofactors are other compounds or ions that enzymes require before their catalytic activity can occur. The protein portion of the enzyme is referred to as the apoenzyme. The enzyme plus the cofactor is known as a holoenzyme. 27/11/2018 Supartono

Cofactors Cofactors may be one of three types Coenzyme: A non protein organic substance that is loosely attached to the enzyme Prosthetic Group: A non protein organic substance that is firmly attached to the enzyme Metal ion activators: K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, or Mo2+, 27/11/2018 Supartono

Types of Cofactors Enzymes have varying degrees of specificity. One cofactor may serve many different enzymes. 27/11/2018 Supartono 15

Types of Cofactors Enzymes have varying degrees of specificity. One cofactor may serve many different enzymes. 27/11/2018 Supartono

Enzymes and Cofactors 27/11/2018 Supartono

Factors Affecting Enzyme Activity

Enzymes and Reaction Rates Factors that influence reaction rates of Enzyme catalyzed reactions include Enzyme and substrate concentrations Temperature pH 27/11/2018 Supartono

Enzymes and Reaction Rates At low concentrations, an increase in substrate concentration increases the rate because there are many active sites available to be occupied At high substrate concentrations the reaction rate levels off because most of the active sites are occupied 27/11/2018 Supartono

Substrate concentration The maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased substrate concentration after this point will not increase the rate.  Vmax is the maximum reaction rate 27/11/2018 Supartono

Substrate concentration Vmax is the maximum reaction rate The Michaelis-Menton constant , Km is the substrate concentration when the rate is ½ Vmax Km for a particular enzyme with a particular substrate is always the same 27/11/2018 Supartono

Effect of Temperature Higher temperature increases the number of effective collisions and therefore increases the rate of a reaction. Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature 27/11/2018 Supartono

Effect of pH Each enzyme has an optimal pH at which it is most efficient A change in pH can alter the ionization of the R groups of the amino acids. When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape. The new shape may not be effective. Pepsin is most efficient at pH2.5-3 while Trypsin is efficient at a much higher pH 27/11/2018 Supartono

pH 27/11/2018 Supartono

Effects of pH on Enzyme Activity Binding of substrate to enzyme Ionization state of “catalytic” amino acid residue side chains Ionization of substrate Variation in protein structure 27/11/2018 Supartono

Temperature 27/11/2018 Supartono

Inhibitors Covalent Non-covalent: reversible Reversible Irreversible 27/11/2018 Supartono

Enzyme Binding Sites Active Site = Binding Site + Catalytic Site Regulatory Site: a second binding site, occupation of which by an effector or regulatory molecule, can affect the active site and thus alter the efficiency of catalysis – improve or inhibit. 27/11/2018 Supartono

Active Site Binding and Catalysis

General Characteristics Three dimensional entity Occupies small part of enzyme volume Substrates bound by multiple weak interactions Clefts or crevices Specificity depends on precise arrangement of atoms in active site 27/11/2018 Supartono

Models Lock and Key Model: the active site exists “pre-formed” in the enzyme prior to interaction with the substrate. Induced Fit Model: the enzyme undergoes a conformational change upon initial association with the substrate and this leads to formation of the active site. 27/11/2018 Supartono

Enzyme Mechanics An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock.  The shape of the enzyme must match the shape of the substrate. Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site. 27/11/2018 Supartono

Induced Fit Theory 27/11/2018 Supartono

Induced Fit Theory The substrate molecule normally does not fit exactly in the active site. This induces a change in the enzymes conformation (shape) to make a closer fit. In reactions that involve breaking bonds, the inexact fit puts stress on certain bonds of the substrate. This lowers the amount of energy needed to break them. 27/11/2018 Supartono

Induced Fit Theory The enzyme does not actually form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape. Because the enzyme does not form chemical bonds with the substrate, it remains unchanged. The enzyme molecule can be reused repeatedly Only a small amount of enzyme is needed 27/11/2018 Supartono 3636

Identification and Characterization of Active Site Structure: size, shape, charges, etc. Composition: identify amino acids involved in binding and catalysis. 27/11/2018 Supartono

Binding or Positioning Site (Trypsin) 27/11/2018 Supartono

Binding or Positioning Site (Chymotrypsin) 27/11/2018 Supartono

Catalytic Site (e.g. Chymotrypsin) 27/11/2018 Supartono

Probing the Structure of the Active Site Model Substrates

Model Substrates (Chymotrypsin) 27/11/2018 Supartono

Peptide Chain? All Good Substrates! 27/11/2018 Supartono

a-amino group? Good Substrate! 27/11/2018 Supartono

Side Chain Substitutions Good Substrates t-butyl- Cyclohexyl 27/11/2018 Supartono

Conclusion Bulky Hydrophobic Binding Site 27/11/2018 Supartono

Probing the Structure of the Active Site Competitive Inhibitors

Arginase 27/11/2018 Supartono

Good Competitive Inhibitors 27/11/2018 Supartono

Poor Competitive Inhibitors All Three Charged Groups are Important 27/11/2018 Supartono

Conclusion Active Site Structure of Arginase 27/11/2018 Supartono

Identifying Active Site Amino Acid Residues

Covalent Inactivation Diisopropyl Phosphofluoridate Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Serine195. Iodoacetic acid inactivates Ribonuclease by reacting with His12 and His119. 27/11/2018 Supartono

Affinity Labeling (General Approach) Positioning Group Reactive Group 27/11/2018 Supartono

Affinity Labeling (Tosyl-L-phenylalanine chloromethylketone) Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Histidine57 27/11/2018 Supartono

Trapping of Enzyme-Bound Intermediate (Chymotrypsin) Implicates Ser195 in catalytic mechanism. 27/11/2018 Supartono

Catalytic Mechanisms

Mechanisms of Catalysis Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation effects Preferential binding (stabilization) of the transition state 27/11/2018 Supartono

Keto-Enol Tautomerization Acid-Base Catalysis Keto-Enol Tautomerization

Uncatalyzed Reaction 27/11/2018 Supartono

General Acid Catalysis 27/11/2018 Supartono

General Base Catalysis 27/11/2018 Supartono

General Acids 27/11/2018 Supartono

General Bases 27/11/2018 Supartono

Ribonuclease A 27/11/2018 Supartono Figure 11-10

Mechanism of RNase A 27/11/2018 Supartono Figure 11-10 part 1

Mechanism of RNase A 27/11/2018 Supartono Figure 11-10 part 2

Covalent Catalysis (Principle) Slow H2O + A–B ——> AOH + BH A-B + E-H ——> E-A + BH E-A + H2O ——> A-OH + E-H Fast 27/11/2018 Supartono

Covalent Catalysis (Chymotrypsin) NOTE: New Reaction Pathway 27/11/2018 Supartono

Metal Ion Catalysis Metalloenzymes: contain tightly bound metal ions for catalytic activity Metal-activated enzymes: loosely bound metal ions from solution Charge stabilization Water ionization Charge shielding 27/11/2018 Supartono

Metalloenzymes Catalytically essential (tightly bound): Fe2+, Fe3+, Cu2+, Mn2+, and Co2+ Structural metal ions: Na+, K+, and Ca2+ Both: Mg2+ and Zn2+ 27/11/2018 Supartono

Proximity and Orientation Effects Rate of a reaction depends on: Number of collisions Energy of molecules Orientation of molecules Reaction pathway (transition state) 27/11/2018 Supartono

Proximity V = k[A][B] [A] and [B] = ~13M on enzyme surface 27/11/2018 Supartono

Biomolecular Reaction of Imidazole with p-Nitrophenylacetate (Intermolecular) 27/11/2018 Supartono Page 336

Intramolecular Rate = 24x Intermolecular Rate Intramolecular Reaction of Imidazole with p-Nitrophenylacetate (Intramolecular) Intramolecular Rate = 24x Intermolecular Rate 27/11/2018 Supartono Page 336

Orientation 27/11/2018 Supartono

Geometry of an SN2 Reaction 27/11/2018 Supartono Figure 11-14

Stabilize Transition State 27/11/2018 Supartono

Steric Strain in Organic Reactions Reaction Rate: R=CH3 is 315x vs R=H 27/11/2018 Supartono Page 338

Effect of Preferential Transition State Binding 27/11/2018 Supartono Figure 11-15

Transition State Analogs Powerful Enzyme Inhibitors

Proline Racemase (planar transition state) 27/11/2018 Supartono Page 339

Transition State Analogs of Proline Binding = 160x versus Proline 27/11/2018 Supartono Page 339

Chymotrypsin Trypsin Elastase etc. Serine Proteases Chymotrypsin Trypsin Elastase etc.

Kinetic Analysis of Chymotrypsin (Hydrolysis of p-nitrophenylacetate) 27/11/2018 Supartono

Mechanism of Chymotrypsin p-nitrophenolate 27/11/2018 Supartono

Special Reactivity of Serine195 Identification of the Catalytic Residues (Reaction of Chymotrypsin with DIPF) Special Reactivity of Serine195 27/11/2018 Supartono

Identification of the Catalytic Residues (Reaction of Chymotrypsin with TPCK) Affinity Labeling 27/11/2018 Supartono

X-Ray Structure of Bovine Trypsin (Ribbon Diagram) 27/11/2018 Supartono

Active Site Residues of Chymotrypsin (Catalytic Triad) 27/11/2018 Supartono Figure 11-26

Catalytic Mechanism of the Serine Proteases Catalytic Triad 27/11/2018 Supartono Figure 11-29 part 1

Catalytic Mechanism of the Serine Proteases 27/11/2018 Supartono Figure 11-29 part 2

Catalytic Mechanism of the Serine Proteases 27/11/2018 Supartono Figure 11-29 part 3

Catalytic Mechanism of the Serine Proteases 27/11/2018 Supartono Figure 11-29 part 4

Catalytic Mechanism of the Serine Proteases 27/11/2018 Supartono Figure 11-29 part 5

Catalytic Mechanism of the Serine Proteases 27/11/2018 Supartono Figure 11-29 part 5

Transition State Stabilization in the Serine Proteases 27/11/2018 Supartono Figure 11-30a

Transition State Stabilization in the Serine Proteases 27/11/2018 Supartono Figure 11-30b

Mechanism of Chymotrypsin p-nitrophenolate 27/11/2018 Supartono