L28-1 Enzyme Catalysis & Biotechnology Bovine Pancreatic RNase A
Biochemistry, Life, and all that L28-2 A brief word about biochemistry traditionally, chemical engineers used organic and inorganic chemistry (along with some physics, lots of math, and other fancy things) to make stuff. Anything bio was regarded as beyond the reach of industrial activities and/or sufficiently uninteresting. This has changed completely in the past few decades. Biochemistry - studies biological (living) species in chemical terms - compositions and structure of biochemical molecules, trying to understand their functions at a molecular level. General observations on biochemical molecules - generally very large: molecular weight ~ O(1,000) O(1,000,000) amu - generally rather simple composition: mostly C, O, H as their main building blocks and relatively few other atoms (such N, P, S) in their functional groups - generally rather complex structures: specific structure is crucial to their highly specialized functions in biological processes. All life processes on earth require energy (processes of bio-molecule synthesis are endothermic), which is obtained indirectly from solar energy through plant photosynthesis.
Photosynthesis and Respiration Photosynthesis and respiration as complementary processes in the living world. Photosynthesis uses the energy of sunlight to produce sugars and other organic molecules. These molecules serve as food for other organisms. Many of these organisms carry out respiration, a process that uses O 2 to form CO 2. In the process, the organisms that respire obtain the chemical bond energy that they need to survive. The first cells on the earth are thought to have been capable of neither photosynthesis nor respiration. However, photosynthesis must have preceded respiration on the earth, since there is strong evidence that billions of years of photosynthesis were required before O 2 had been released in sufficient quantity to create an atmosphere rich in this gas. (The earth's atmosphere presently contains 20% O 2.) L28-3
Maintaining Order Cells must maintain highly organized, low-entropy state at the expense of free energy. Cells cannot use heat for energy (cells are very heat-sensitive!). Energy released in exergonic reactions used to drive endergonic reactions. Require energy released in exergonic reactions (ATP) to be directly transferred to chemical-bond energy in the products of endergonic reactions. Endergonic/exergonic refer to free energy changes (ΔG). Endothermic and exothermic refer to ΔH. For many chemical reactions, entropy contributions are relatively small, so chemists usually refer to ΔH. For many biological reactions, entropy contributions are significant, so biochemists usually talk about ΔG. L28-4
ATP Major Energy Carrier Formation of ATP requires the input of a large amount of energy, stored in the bond energy by joining P i to ADP. This energy released when ATP converted to ADP and P i. ATP is the universal energy carrier of the cell. ΔH ΔH ~ ~ 30 30 kj/mol kj/mol + H 2 O L28-5
Metabolism: Glycolysis A bag of sugar can be stored for years with little conversion to CO 2 and H 2 O However: : this conversion is basic to life -> > need to accelerate it! Mother Nature s Solution: Glycolysis the break-down of glucose to pyruvate, catalyzed by enzymes Embden-Meyerhof Pathway: universal pathway - occurs in essentially all organisms overall net gain of 2 ATP L28-6
L28-7 The 10 Steps of Glycolysis I
L28-8 The 10 Steps of Glycolysis - II
L28-9 The 10 Steps of Glycolysis - III
Enzymes: Why? L28-10 Living organisms must be able to carry out chemical reactions which are thermodynamically highly unfavorable Break and form covalent bonds Move large structures Make complex three dimensional structures Regulate gene expression They do so through enzyme catalysis. Enzymes have have immense importance in in a wide variety of of fields: Genetic Genetic diseases diseases are are frequently frequently defects defects in in enzymes enzymes or or increased/decreased levels levels of of enzymes enzymes Many Many modern modern drugs drugs exert exert effects effects by by interacting interacting with with enzymes enzymes Used Used in in food food processing processing and and in in chemical chemical industry industry Enzyme Enzyme inhibitors inhibitors are are a a foundation foundation of of biological biological weapons weapons (as (as well well as as of of some some of of the the counter-measures) counter-measures)
Enzymes, Proteins and Amino Acids Enzymes are proteins that act as catalysts for biochemical reactions ions. Proteins are very large biomolecules present in living cells (~50% dry wt of our body). All proteins are composed of the same building blocks - α-amino acids. non-ionic form H R O H 2 N-C-C-OH H H 2 N-C-C-OH R O or H O + H 2 N-C-C-OH = R H + H 3 N-C-C-O - R O zwitter-ion (at neutral ph) α-amino acids are linked by amide groups, which are formed in a condensation reaction between the acid and amine groups of two amino acids: H R O H 2 N-C-C-N-C-C-OH H H R O amide group + H 2 O L28-11 This bond is also referred to as peptide bond, and the resulting molecules are peptides.
Formation of a Peptide Bond general reaction scheme: example: Peptides, Polypeptides, and Proteins (among them: Enzymes) are amino acid polymers! L28-12 Amino Amino Acid Acid Peptide Poly-Peptide Protein Protein (e.g. (e.g. Enzymes)
L28-13 The 20 Amino Acids Found in Proteins
Names & Types of Enzymes L28-14 Enzyme names (mostly) end in ase - identifies a reactant: sucrase - reacts sucrose, lipase - reacts lipid - common names of digestion enzymes still use in: pepsin, trypsin - describes function of enzyme: Class Class Oxidoreductases Transferases Hydrolases Lyases Lyases Isomerases Ligases Ligases Reactions catalyzed
Learning Check E1 Match the type of reaction with the enzymes: (1) aminase (2) dehydrogenase (3) Isomerase (4) synthetase ( ) Converts a cis-fatty acid to trans. ( ) Removes 2 H atoms to form double bond ( ) Combine two molecules using ATP ( ) Adds NH 3 L28-15
Enzyme Structure The three -dimensional structure of enzymes is crucial for their functionality. Four hierarchical levels of enzyme structure are distinguished: primary, secondary, tertiary and quaternary. L28-16
Enzymes: Primary Structure Primary structure: sequence of amino acids: KVFGRCELAAAMKRHGLDNY methionine (M) phenylalanine (F) lysine (K) isoleucine (I) arginine (R) 3 + + 2 tyrosine (Y) valine (V) histidine (H) tryptophan (W) leucine (L) glycine (G) alanine (A) aspartic acid (D) aspargine (N) serine (S) cysteine glutamic acid (E) glutamine (Q) threonine (T) proline L28-17
L28-18 Primary Structure of Bovine Insulin First protein to be fully sequenced; Fred Sanger, 1953. For this, he won his first Nobel Prize (his second was for DNA sequencing).
Enzymes: Secondary Structure Secondary structure: packing of amino acids (helix, sheet), i.e. the spatial arrangement of the back-bone of the enzyme (without special consideration of side groups). Alpha-Helix Beta-Sheet L28-19
L28-20 20 Enzymes: Tertiary Structure Tertiary structure: cross-linking and 3D conformation, i.e. complete spatial arrangement of one enzyme alpha-helix beta-sheet ribonuclease A loop (non-repeating coil structure)
L28-21 21 Enzymes: Quaternary Structure Quaternary structure: enzyme oligomers, i.e. spatial arrangement of enzymes (and other peptides) which consist of several subunits.
Enzyme Structure: Recap L28-22 22 Four hierarchical levels of enzyme structure: Primary structure: sequence of of amino amino acids acids (1D) (1D) Secondary structure: spatial spatial arrangement of of backbone (2D) (2D) Tertiary structure: detailed spatial spatial conformation of of one one enzyme (3D) (3D) Quaternary structure: spatial spatial conformation of of multiple enzymes ( oligomers )
Enzyme Action: Models L28-23 23 Lock and Key Model An enzyme binds a substrate in a region called the active site Only certain substrates can fit the active site Amino acid R groups in the active site help substrate bind Induced Fit Model Enzyme structure flexible, not rigid Enzyme and active site adjust shape to bind substrate Increases range of substrate specificity Shape changes also improve catalysis during reaction -> transition-state like configuration In each case, an enzyme-substrate complex is formed, the respective bonds in the substrate are formed or broken (i.e. the reaction occurs), and the product(s) are released: E + S <=> ES <=> E + P
Lock and Key Model L28-24 24 (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can make noncovalent bonds only with certain ligands. (B) Close-up view of an actual binding site showing the hydrogen bonds and ionic interactions formed between a protein and its ligand (in this example, cyclic AMP is the bound ligand).
L28-25 25 Induced Fit Model Induced Conformational Change in Hexokinase
L28-26 26 Enzyme-Substrate Interaction