CHAPTER Biochemistry Enzymes

2 Metabolic pathways
§  Series of reactions that convert subtrates to
Catalysts that accelerate chemical reactions
§  Enzymes have three properties
Chemistry, products 1. Active at low concentrations
Biochemistry, and Cell §  Catalyzed by enzymes
2. Increase the rate of reactions but are not altered
Physiology §  Synthesis (anabolic)
Part 2 3. Do not change the products
§  Degradative (catabolic)
§  Most are made of proteins
§  Metabolic pathways are linked by intermediates
§  Some are made of RNA (ribozymes)
§  Metabolism – sum of metabolic pathways for the
synthesis and breakdown of molecules.
PowerPoint® Lecture Slides prepared by
Stephen Gehnrich, Salisbury University

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Cofactors Reaction Acceleration Reaction Acceleration

§  Nonprotein components of enzymes §  Enzymes accelerate reactions by reducing

§  Many are loosely associated with enzymes activation energy (EA)
§  Same substrate and product as uncatalyzed reaction
§  Prosthetic group – cofactor covalently bonded into
the enzyme §  Enzyme catalyzed reaction has a different
intermediate at the transition state
§  Coenzymes – organic cofactors usually derived §  S + E ↔ ES ↔ ES* ↔ EP* ↔ EP ↔ E + P
from vitamins
§  Active site – location in enzyme where substrate
§  Inorganic ion cofactors – copper, iron, magnesium, binds
zinc §  Specific, 3-dimensional shape

Figure 2.12
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 Enzyme Kinetics Michaelis-Menten Rectangular Hyperbola Sigmoidal Relationships

Conditions that influence the rate of enzymatic v = Vmax × [S] / ([S] + Km) §  Homotropic enzymes – sigmoidal relationship
reactions §  v = initial velocity between v and [S]
§  For example, changing the concentration of §  Vmax = maximum velocity §  Cooperativity – enzymes show increased affinity
substrate and product will affect reaction rates for S with increasing [S]
§  Km = indicator of affinity of enzyme for the
substrate §  Hill coefficient – degree of cooperativity (slope at
inflection)

Figure 2.13 Figure 2.14

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Hyperbolic vs. Sigmoidal Relationships Environmental Effects Environmental Effects

Enzyme activity is affected by temperature, pH,

salinity, and hydrostatic pressure
Enzymes are affected in different ways
§  Changes in weak bonds alter three-dimensional
structure
§  Changes in ionization state of amino acids within
the active site
§  Changes in the ability of the enzyme to undergo
structural changes necessary for catalysis

Figure 2.13 and Figure 2.15 Figure 2.16

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 Evolutionary Adaptation of Enzymes Regulation of Enzyme Activity Regulation of Enzyme Activity

Enzymes have evolved to meet physiological Other molecules can affect enzyme kinetics
conditions §  Competitive inhibitors
§  Block the active site
§  Allosteric regulators
§  Alter the three-dimensional shape of the enzyme
§  Covalent modification
§  Phosphorylation alters enzyme activity

Figure 2.17 Figure 2.18

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Energy Storage Reducing Energy High Energy Bonds

Cells store energy in two main forms §  Reducing equivalents – electrons bound to a carrier Energy can be “stored” in covalent bonds
§  Example: NADH, NADPH, FADH2, FMNH2
§  Reducing energy §  Energy is released when bonds are broken
§  Oxidoreductases – enzymes that transfer reducing
§  High energy bonds equivalents between reduced (energy-rich) and oxidized §  ATP is the most common “high energy” molecule
(energy-poor) molecules
§  Example: lactate dehydrogensae
§  NAD+ + lactate → NADH + H+ + pyruvate
§  Redox status – reducing energy within a cell
§  reduced form/oxidized form
§  Example: [NADH/NAD+]

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 High Energy Bonds Biomolecules Proteins

Four main types §  Contribute to cell structure and function

§  Proteins §  Mediate all cellular processes
§  Carbohydrates §  Enzymes
§  Lipids §  Have complex three-dimensional structure
§  Nucleic acids §  Protein structure is coded in DNA

Figure 2.19
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Amino Acids Protein Structure Primary Structure

§  Proteins are polymers of amino acids Linear sequence of amino acids joined by covalent
§  Amino acids – amino group (–NH2) and carboxylic bond between the carboxyl and amino group
acid group (–COOH) §  Peptide bond
§  Termed α-amino acids because –NH2 and –COOH
are located on the first (α) carbon
§  Distinguished by side groups (R)
§  Can be nonpolar (hydrophobic), polar-uncharged
(hydrophilic) and polar-charged (hydrophilic)

Figure 2.20
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 Secondary Structure Tertiary Structure Quaternary Structure

§  Localized folding Covalent bonds Protein made of multiple polypeptide chains
§  Linked by hydrogen bonds §  Disulfide bonds §  Dimer – two subunits
§  α-helix Weak bonds §  Homodimer – identical proteins
§  β-sheet §  Heterodimer – different proteins
§  van der Waals forces
§  Ionic bonds §  Trimer – three subunits
§  Hydrogen bonds §  Tetramer – four subunits

Figure 2.21 Figure 2.22

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Protein Structure Molecular Chaperones Carbohydrates

Proteins function properly only when folded into §  “Hydrates of carbon”
correct three-dimensional shape §  Many hydroxyl (–OH) groups
§  Some proteins fold spontaneously §  Glucose is the most common carbohydrate in
§  Some are helped by molecular chaperones animal diets
§  Force protein into conformation that allows weak §  Energy metabolism
bonds to form §  Biosynthesis – precursor to many other
§  For example, heat shock proteins carbohydrates

Figure 2.20
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 Monosaccharides Monosaccharides Disaccharides

§  Used for energy and biosynthesis §  Two monosaccharides connected by a covalent
§  Small carbohydrates have three to seven carbons – bond
six is most common §  Bond is broken during metabolism

Figure 2.23
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Disaccharides Carbohydrates + Other Macromolecules Complex Carbohydrates

Glycosylation – addition of carbohydrates to other §  Polysaccharides

macromolecules §  Long chain of monosaccharides
Alters function of the macromolecule §  Energy storage
§  Example: glycogen, starch
For example, glycolipids, glycoproteins
§  Structural molecules
§  Both are typically found in plasma membranes and §  chitin, hyaluronate, cellulose (in plants)
extracellular fluid

Figure 2.23
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 Complex Carbohydrates Glycogen Metabolism Glucose Metabolism

Glycogen synthesis (glycogenesis) Glucose synthesis (gluconeogenesis)

Glycogen breakdown (glycogenolysis) §  Uses reducing equivalents
§  Requires energy
§  2 pyruvate + 4ATP + 2GTP + 2NADH + 4H2O à
glucose + 4ADP + 2GDP + 6Pi + 2NAD+ + 2H+

Figure 2.25a Figure 2.26

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Gluconeogenesis Glucose Metabolism Glycolysis

Glucose breakdown (glycolysis)

§  Produces reducing equivalents
§  Releases energy
§  Glucose + 2ADP + 2NAD+ à 2ATP + 2 pyruvate +
2NADH + 2H+
§  Takes place in cytoplasm
§  Does not require oxygen
§  Produces intermediates for synthesis of various molecules
§  Carbohydrates, nucleic acids, amino acids, and fatty acids

Figure 2.27 Figure 2.28

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 Oxidation of Pyruvate in the Presence of O2 Oxidation of NADH in the Presence of O2 Redox Shuttles

Glycolysis Glycolysis can only continue if NADH is

§  Converts carbohydrates to pyruvate within the oxidized to NAD+
cytoplasm
Two “redox shuttles” carry reducing equivalents
§  Lactate and amino acids can also be converted to
from cytoplasm into mitochondria
pyruvate
§  Pyruvate is carried into the mitochondria §  α-glycerophosphate shuttle
Pyruvate dehydrogenase (PDH) §  Malate-aspartate shuttle
§  Pyruvate is oxidized by PDH to form acetyl CoA +
NADH

Figure 2.29
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Oxidation of NADH in the Absence of O2 Oxidation of NADH in the Absence of O2 Lipids

§  NADH cannot be used by mitochondria when oxygen is not §  All are hydrophobic (do not dissolve in water)
present
§  Carbon backbone
§  NADH is oxidized in the cytoplasm
§  Linear – aliphatic
§  pyruvate + NADH + H+ à lactate + NAD+
§  Catalyzed by the enzyme lactate dehydrogenase (LDH) §  Ring – aromatic
§  Other anaerobic pathways form less toxic end products and §  Examples: fatty acids, triglycerides, phospholipids,
more ATP than lactate (2 ATP) steroids
§  For example, succinate (4 ATP) and proprionate (6 ATP) §  Lipids are used for energy metabolism, cell
structure, and signaling

Figure 2.30
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 Fatty Acids Fatty Acids Fatty Acid Synthesis

§  Chain of carbon atoms ending with a carboxyl Fatty acids are synthesized from Acetyl CoA
group §  Catalyzed by the enzyme fatty acid synthase (FAS)
§  Usually an even number of carbons
§  Saturated
§  No double bonds between carbons
§  Unsaturated
§  One or more double bonds between carbons

Figure 2.31
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Fatty Acid Oxidation (β-Oxidation) Fatty Acid Oxidation (β-Oxidation) Ketones

Breakdown of fatty acids §  Some tissues cannot metabolize fatty acids, but
§  β-oxidation they can metabolize ketones
§  For example, vertebrate brain, shark muscle
§  Takes place in mitochondria
§  Results in formation of Acetyl CoA §  Ketogenesis
§  Fatty acids (acetyl CoA) are converted to ketones
§  Acetyl CoA is oxidized
§  Ketolysis
§  Ketones are broken down to acetyl CoA

Figure 2.32
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 Ketone Metabolism Triglycerides Triglycerides

§  Fatty acids esterified to a glycerol backbone

§  For example, mono-,di-, tri-acylglycerol
§  Fatty acids are stored as triglycerides
§  Long-term storage of fatty acids
§  Primary storage tissues: adipose and liver
(vertebrates), hepatopancreas (invertebrates)
§  Lipases break the bond between fatty acid and
glycerol backbone (lipolysis)

Figure 2.33 Figure 2.34

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Triglyceride Synthesis or Lipogenesis Phospholipids Phosphoglycerides

§  Dominate biological membranes

§  Two classes of phospholipids in animal cells:
§  Phosphoglycerides
§  Constructed from diacylglycerol
§  Polar group on third carbon
§  Sphingolipids
§  Sphingosine backbone
§  Phospholipids are broken down by phospholipases

Figure 2.35 Figure 2.36a

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 Sphingolipids Steroids Mitochondrial (Oxidative) Metabolism

§  Four hydrocarbon rings Energy-yielding reactions that require oxygen

§  Synthesis involves §  Enzymes convert nutrients into metabolites
many intermediates §  Metabolites enter mitochondria
§  Many metabolites are converted to acetyl CoA
§  Acetyl CoA enters the tricarboxylic acid cycle (TCA cycle)
§  Acetyl CoA is oxidized to form reducing equivalents
§  Reducing equivalents are oxidized to release energy

Figure 2.36b Figure 2.37

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Formation of acetyl CoA Oxidative Metabolism Tricarboxylic Acid (TCA) Cycle

Acetyl CoA §  Generates reducing equivalents within the

↓ mitochondria
Tricarboxylic acid (TCA) cycle §  Acetyl CoA + 3NAD+ + GDP + Pi + FAD à 2CO2 +
acetyl CoA à CO2 + reducing equivalent (NADH and 3NADH + FADH2 + GTP
FADH2) and GTP
§  Amphibolic pathway
↓
§  Some intermediates are broken down (catabolic)
Electron transport system (ETS)
§  Some intermediates are used for syntheses (anabolic)
reducing equivalents are oxidized to release energy
↓
Oxidative phosphorylation
ATP synthesis (phosphorylation)

Figure 2.38
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 Tricarboxylic Acid (TCA) Cycle Electron Transport System (ETS) Electron Transport System (ETS)

Electrons from NADH and FADH2 are transferred to

the ETS
§  Found within the inner mitochondrial membrane
§  Composed of four multisubunit proteins
(complexes I, II, III, IV) and two electron carriers
(ubiquinone and cytochrome c)
§  Oxidation: 4e– + 4H+ + O2 à 2H2O
§  Generates a proton gradient, heat, water, and
reactive oxygen species (ROS)

Figure 2.39 Figure 2.40

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ATP Synthesis Phosphocreatine Phosphocreatine

§  Phosphorylation: ADP + Pi à ATP §  Alternative high-energy phosphate compound

§  Proton motive force (Δp) §  Creatine + ATP ↔ ADP + phosphocreatine
§  pH gradient and the membrane potential (ΔΨ) §  Creatine phosphokinase (CPK)
§  F1F0ATPase uses energy in Δp to produce ATP §  Reaction is reversible so phosphocreatine can be
§  There is no physical linkage between oxidation and used to produce ATP when levels are low
phosphorylation
§  Two processes are functionally coupled through Δp

Figure 2.41
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 Integration of Metabolic Pathways Reciprocal Regulation Respiratory Quotient

§  Fluctuations in nutrient availability, energy Type of fuel being used can be monitored by
demand, and environmental conditions measuring the RQ
§  Reciprocal regulation avoids simultaneous §  Respiratory quotient (RQ) = CO2 production/O2
synthesis and degradation (futile cycles) consumption
§  Use of appropriate metabolic “fuel” §  RQ – 0.7 for lipids, 1.0 for carbohydrates
§  Carbohydrate vs. lipid
§  Energetic intermediates regulate balance between
anabolism and catabolism

Figure 2.42
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