Enzymes and Reaction Rate

Enzymes

• Many enzymes sufficiently fold into the correct structure as a polypeptide
• Enzymes bind substrate molecules at active sites to form an enzyme-substrate complex, leading to products
• Coenzymes are organic molecules like vitamins, and cofactors are metal ions
• An enzyme needing coenzymes or cofactors that are absent is called an apoenzyme and lacks biological function
• An apoenzyme is biologically active once coenzymes or cofactors are added and becomes a holoenzyme
• Enzymes raises reaction rates without affecting equilibrium

Free Energy and Reaction Rate

• Free energy changes occur as molecules go from substrate to product, indicating equilibrium
• Reaction rate relies on energy from the transition state, which is the highest energy moment
• Enzymes lower free energy at the transition state, which accelerates reaction rates
• Enzymes lower free energy via binding and chemical effects

Binding Effects

• Substrate binding involves interaction with the enzyme and the substrate
• Water molecules are stripped away
• Entropy is lessened
• Molecules come together to a reactive position
• Induced fit and changes in the conformation of the substrate occur
• Transition state stabilization means an active site is complementary to the substrate, which ensures specificity and changing substrate to the transition state
• Enzymes possess high affinities for transition states over substrates
• Transition state analogs bind to the enzyme's active site with very high affinity and are useful as competitive inhibitors

Chemical Effects

• Acid/base catalysis involves enzymes picking up or donating protons, usually with histidine residues
• Covalent catalysis creates a covalent linkage
• A covalent linkage formation occurs in two stages: forming a covalent bond on substrate molecule to break into 2, and regeneration of the free enzyme
• Serine proteases show both acid/base and covalent reactions

Enzyme Kinetics

• Michaelis-Menten plot shows velocity vs. substrate concentration
• Vmax is the point where velocity stops depending on substrate concentration
• Km measures substrate concentration at 1/2 Vmax
• Formula: Vo = Vmax[S]/([S] + Km)
• Lineweaver-Burk plots are more accurate because they are a double reciprocal representation
• Vertical axis is 1/Vmax
• Horizontal axis is -1/Km

Reversible Inhibitors

• Competitive inhibitors compete with the substrate while binding to the active site
• Competitive inhibitors are only able to bind free enzymes
• Adding excess substrate washes out competitive inhibitors
• Because additional substrate is needed to reach Vmax or more Vmax, the Km is increased
• Uncompetitive inhibitors only bind the enzyme-substrate complex and their binding causes a conformational change creating a binding site for the inhibitors
• The equation V = [ES]K2 shows velocity depends on the ES complex concentration multiplied by the K2 rate constant
• A decrease in the ES complex velocity also decreases ES complex concentration
• More E and S must bind to establish equilibrium, and the enzyme's affinity for the substrate has increased
• All of this corresponds to a decrease in Km
• Non-competitive inhibitors can bind free enzymes or the enzyme-substrate complex
• These inhibitors do not change the affinity of the enzyme for the substrate, and decreasing the ES complex concentration decreases Vmax

Serine Proteases

• Serine proteases cleave polypeptide chains uniquely
• Trypsin cleaves beside positive residues
• Chymotrypsin cuts beside aromatics
• Elastase cuts beside small hydrophobic residues (alanine, glycine)
• Serine proteases use a catalytic triad to cut peptides
• Chymotrypsin showcases acid-base and covalent catalysis

Chymotrypsin Stages

• Stage 1: Histidine extracts a proton as a base from serine's hydroxyl, which activates the oxygen to attack the carbonyl carbon to cleave the peptide
• Stage 2: Histidine acts as an acid to donate the protein to amide nitrogen and cuts the substrate in 2
• Stage 1: Histidine acts as a base by extracting a proton molecule from water, which is used to attack the enzyme and the substrate at the covalent linkage point
• Stage 2: Histidine acts as an acid so it can donate a proton and reform the serines hydroxyl group

Regulation of Enzyme Activity

• Enzyme activity is controlled long term through availability
• Activity is regulated short term
• Activity regulation via controlled availability of enzyme, by induced production or targeted destruction of enzymes
• Activity regulation via covalent modification - phosphorylation
• Phosphoryl groups are added by kinases
• Phosphoryl groups are reversibly added removed by phosphatases

Glycogen Regulation

• Phosphorylation of both enzymes produces a catalase enzyme
• When unphosphorylated, both enzymes produce an anabolic enzyme
• Enzymes create glycogen from glucose residues, and is catalyzed with glucose synthase, which is anabolic
• Presence of insulin drives glucose storage as glycogen
• Catabolic glucose phosphorylase catalyses glycogen transforming into glucose
• During hunger or fear both enzymes phosphorylate in the presence of epinephrine or glucagon

Futile Cycling

• Futile cycling occurs when catabolic and anabolic processes operate simultaneously
• This is avoided by reciprocal regulation
• Enzyme activity is regulated via non-covalent or allosteric regulation

Allosteric Enzymes

• Allosteric enzymes possess multiple binding sites
• Active sites bind the substrate molecules
• Allosteric sites bind allosteric modulators
• Allosteric enzymes tend to have quaternary structures that are large and complex
• Allosteric enzymes have 2 conformations and exist in either an R or T state
• Modulators influence equilibrium either to the R or T state
• Allosteric enzymes tend to be slow, act as rate-limiting steps, and catalyse committed steps
• Feedback is regulated from the final product through negative feedback
• Allosteric enzymes show sigmoidal relationships and do not obey the Michaelis-Menten kinetics
• Their oxygen binding curves are similar to hemoglobin's
• Curve shape of curve allows sensitivity to substrate concentration changes and a threshold effect means small concetaitons affect velocity

Glycolysis

• Glycolysis converts glucose into ATP
• PFK1 includes the allosteric inhibitor PEP and the allosteric activator ADP
• Relative activity relies on PEP and ADP concentrations
• When ADP > PEP the activity is fastest
• When PEP > ADP the activity is slowest

Carbohydrates

• Carbohydrates are hydrates of carbon as their molecular formula shows every carbon with an associated water molecule
• There are multiple chiral carbons present
• Formula 2^n determines the stereoisomers, and n is the chiral carbons present
• When naming L or D sugars, look at the chiral carbon furthest from the carbonyl carbon
• Differing chiral carbons are epimers
• Carbohydrates are ketoses or aldoses
• Five-carbon sugars are ribose
• Six-carbon sugars are glucose, fructose, and galactose
• Larger carbohydrates form cyclic structures

Properties of Sugar

• Hydroxyl groups attach to the carbonyl carbon
• A non-chiral carbon converts to a chiral carbon
• Cyclization causes the carbon to become a anomeric
• C1 is for Aldoses
• C2 is for ketoses
• At the anomeric carbon are the isomers alpha and beta
• Alpha and beta anomers are versions of each other
• Mutarotation is the interconversion of the alpha and beta forms through a linear intermediate

Disaccharides

• To name disaccharides two, six-carbon aldoses link together in a pyran ring structure
• Disaccharides contains either glucose or galactose
• Presence of glucose or galactose is determined on molecule structure
• OH up shows galactose
• OH down shows glucose
• Having a carbon with a free anomeric carbon means it is a reducing end

Polysaccharides

• Homopolysaccharides are one type of monosaccharide
• Heteropolysaccharides are multiple types of monosaccharides
• Plants use starch for energy storage
• Amylose is unbranched
• These contain a glucose residue linked through a(1-4) linkages
• Amylopectin is branched like amylase but it contains branche a(1-6) tides every 24-30 residues

Glycogen

• Glycogen is a(1-4) + a(1-6) and contain greater frequency of branach
• Having more branch points allows for more non-reducing ends and accelerates mobilization of glucose
• Structural polysaccharides use beta linkages as opposed to alpha linkages

Lipids

• Lipids are united by aggregates
• Fatty acids contain a carboxyl group followed by a hydrocarbon tail
• These differ in the length of hydrocarbon tails and location of double bonds
• Tails contain 12-24 carbons in length with an even number of carbons
• No double bonds demonstrates saturation
• One double bond causes unsaturation
• Multiple double bonds means polyunsaturation

Properties of Fatty Acids

• A long saturated fatty acid has a greater chance of being a solid
• The number of carbons, number of double bonds, and location of the double bonds determines a fatty acid
• Lipids store energy with triacylglycerol through ester linkages with 3 fatty acids and a glycerol backbone - ester linkage between the hydroxyl group of glycerol and the carboxyl group of the fatty acids
• Advanced energy storage demonstrates a low oxidation and hydration state
• Hydrophobicity creates additional energy in a gram-to-gram measurement
• Saponification separates fatty acids from ester linkages through base treatment

Membrane Lipids and fat Soluble Vitamins

• Membrane lipids contain 2 hydrocarbon tails and polar heads
• Backbone differs in glycerol or sphingosine, a long chain amino alcohol to which one covalently links a fatty acid
• Differences specialized polar head groups
• Fat-soluble vitamins include
• Vitamin D is for bone formation
• Vitamin A is for vision
• Vitamin E neutralizes free radicals
• Vitamin K is for coagulation

Cholesterol

• Cholesterol are planar rings coming together and are bulky groups
• It mediates membrane fluidity
• It serves as a precursor for active signaling molecules, like sex hormones and corticosteroids

Eicosanoids

• Eicosanoids are paracrine hormones that act close to their production site
• A few are prostaglandins, which cause fever and inflammation
• Thromboxanes cause blood clot formation
• Leukotriene causes smooth is smooth muscle contraction
• Aspirin blocks production of prostaglandin and thromboxane but influence production of Leukotriene

Membranes

• Membranes undergo specializations in lipids and carbohydrates
• Membranes are asymmetric
• Membranes are made of lipids, protein, and their relative concentration determines function (more active = more protein)

Properties of Membranes

• The Fluid mosaic model refers to membranes that are held together by non-covalent forces allowing freedom of movement within the membrane's plane
• Peripheral membrane proteins are associated across the membrane's face through hydrogen bonds or electrostatic interaction
• Attached hydrocarbon tails that are covalently linked on lipid-linked proteins mean there is a single thyrogen inside the cell, and GPI anchors that associate with the lid racks exist on the outside of the cell
• Integral membrane-spanning regions are hydrophobic residues
• Sequencing a protein can identify membrane-spanning regions and determine if it is a transmembrane protein

Lipid Racks

• Lipid racks contain bulges of longer hydrocarbon tails that are coming together
• Having longer hydrogens in Sphinglpidis stabilizes these racks
• Process is spontaneous

Getting Molecules Across the Membrane

• Unassisted Diffusion transports small nonpolar molecules across the membrane
• Facilitated diffusion has channels and carriers, the latter binds to a molecule in order to transport it
• Active transport involves energy and include:
  • Primary Active Transport using ATP
  • P-Type: phosphorylated intermediate
  • V-type: pump proteins into vesicles
  • ABC transporters: pump toxins out
• Secondary Active Transport uses the gradient of molecules as a source of energy, like epithelial cells taking in glucose as sodium moves down its gradient and glucose into the intestinal cells

Nucleic Acids

• The different nitrogenous bases must be uniquely identifiable
• Nucleosides include different nitrogenous bases, and also differ depending on the presence of a hydroxyl group, or the presence of phosphoryl groups on the 5’ carbon of the ribose
• DNA and RNA strands are formed the same way
• Phosphodiester Linkages exists from the 5’-3’ carbons of 2 different nucleotide molecules (each has as negative charge with it)
• Nitrogenous bases are linked to the 1’ carbon of the ribose

RNA and DNA

• DNA and RNA's varying stabilities is due to the presence of a hydroxyl group on the 2’ carbon making RNA susceptible to base hydrolysis
• Nucleic acid strands adopt higher order structures and interact with nonpolar nitrogenous bases
• The hydrogen bonds are optimized in the nitrogenous bases
• Complementary, antiparallel strands come together in classic Watson-Crick base pairs

Chargoffs Rule

• Chargoffs rule combines a purine with a pyrimidine (AT, GC)
• Eukaryotic Duplex DNA requires histones for packaging
• Histones are cationic proteins with positive charges that are highly conserved

Nucleosomes Cores

• 8 polypeptide (H2A, H2B, H3, H4) molecules wrap 146 base pairs
• Another histone, H1, binds to the stretch of DNA
• A mix of electrostatic reactions drives these interactions

Chromosome Structure

• Eukaryotic chromosomes are linear, whereas bacterial DNA is circular
• Telomeric sequences (repeated sequences) stop against the continuous loss of DNA
• Introns (intervening sequences) get spliced so there is a functional mRNA molecule
• Restriction enzymes exist in prokaryotic cells and recognize and cut specific DNA sequences
• DNA sequences tend to be palindromic, and useful for restriction fragment length polymorphism analysis in forensics
• PCR denatures strands and then anneals them (cools to allow strands to come back together)
• A heat stable polymerase multiplies and amplifies specific regions of DNA