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ACS BIOCHEMISTRY EXAM Question and
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Henderson-Hasselbach Equation - SOLUTION pH = pKa + log ([A-] / [HA]) FMOC Chemical Synthesis - SOLUTION Used in synthesis of a growing amino acid chain to a polystyrene bead. FMOC is used as a protecting group on the N-terminus. Salting Out (Purification) - SOLUTION Changes soluble protein to solid precipitate. Protein precipitates when the charges on the protein match the charges in the solution. Size-Exclusion Chromatography - SOLUTION Separates sample based on size with smaller molecules eluting later. Ion-Exchange Chromatography - SOLUTION Separates sample based on charge. CM attracts +, DEAE attracts -. May have repulsion effect on like charges. Salt or acid used to remove stuck proteins. Hydrophobic/Reverse Phase Chromatography - SOLUTION Beads are coated with a carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile). Affinity Chromatography - SOLUTION Attach a ligand that binds a protein to a bead. Elute with harsh chemicals or similar ligand. SDS-PAGE - SOLUTION Uses SDS. Gel is made from cross-linked polyacrylamide. Separates based off of mass with smaller molecules moving faster. Visualized with Coomassie blue. SDS - SOLUTION Sodium dodecyl sulfate. Unfolds proteins and gives them uniform negative charge.
Isoelectric Focusing - SOLUTION Variation of gel electrophoresis where protein charge matters. Involves electrodes and pH gradient. Protein stops at their pI when neutral. FDNB (1-fluoro-2,3-dinitrobenzene) - SOLUTION FDNB reacts with the N- terminus of the protein to produce a 2,4-dinitrophenol derivative that labels the first residue. Can repeat hydrolysis to determine sequential amino acids. DTT (dithiothreitol) - SOLUTION Reduces disulfide bonds. Iodoacetate - SOLUTION Adds carboxymethyl group on free -SH groups. Blocks disulfide bonding. Homologs - SOLUTION Shares 25% identity with another gene Orthologs - SOLUTION Similar genes in different organisms Paralogs - SOLUTION Similar "paired" genes in the same organism Ramachandran Plot - SOLUTION Shows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α- helices. Glycine Ramachandran Plot - SOLUTION Glycine can adopt more angles. (H's for R-group). Proline Ramachandran Plot - SOLUTION Proline adopts fewer angles. Amino group is incorporated into a ring. α-helices - SOLUTION Ala is common, Gly & Pro are not very common. Side-chain interactions every 3 or 4 residues. Turns once every 3. residues. Distance between backbones is 5.4Å. Helix Dipole - SOLUTION Formed from added dipole moments of all hydrogen bonds in an α-helix. N-terminus is δ+ and C-terminus is δ-. ß-sheet - SOLUTION Either parallel or anti-parallel. Often twisted to increase strength.
Temperature Denaturation of Protein - SOLUTION Midpoint of reaction is Tm. Cooperative Protein Folding - SOLUTION Folding transition is sharp. More reversible. Folding Funnel - SOLUTION Shows 3D version of 2D energy states. Lowest energy is stable protein. Rough funnel is less cooperative. Protein-Protein Interfaces - SOLUTION "Core" and "fringe" of the interfaces. Core is more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic. π-π Ring Stacking - SOLUTION Weird interaction where aromatic rings stack on each other in positive interaction. σ-hole - SOLUTION Methyl group has area of diminished electron density in center; attracts electronegative groups Fe Binding of O2 - SOLUTION Fe2+ binds to O2 reversible. Fe3+ has an additional + charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments. Ka for Binding - SOLUTION Ka = [PL] / [P][L] ϴ-value in Binding - SOLUTION ϴ = (bound / total)x100% ϴ = [L] / ([L] + 1/Ka) Kd for binding - SOLUTION Kd = [L] when 50% bound to protein. Kd = 1/Ka High-Spin Fe - SOLUTION Electrons are "spread out" and result in larger atom. Low-Spin Fe - SOLUTION Electrons are less "spread out" and are compacted by electron rich porphyrin ring. T-State - SOLUTION Heme is in high-spin state. H2O is bound to heme. R-State - SOLUTION Heme is in low-spin state. O2 is bound to heme.
O2 Binding Event - SOLUTION O2 binds to T-state and changes the heme to R-state. Causes a 0.4Å movement of the iron. Hemoglobin Binding Curve - SOLUTION 4 subunits present in hemoglobin that can be either T or R -state. Cooperative binding leads to a sigmoidal curve. Binding Cooperativity - SOLUTION When one subunit of hemoglobin changes from T to R-state the other sites are more likely to change to R- state as well. Leads to sigmoidal graph. Homotropic Regulation of Binding - SOLUTION Where a regulatory molecule is also the enzyme's substrate. Heterotropic Regulation of Binding - SOLUTION Where an allosteric regulator is present that is not the enzyme's substrate. Hill Plot - SOLUTION Turns sigmoid into straight lines. Slope = n (# of binding sites). Allows measurement of binding sites that are cooperative. pH and Binding Affinity (Bohr Affect) - SOLUTION As [H+] increases, Histidine group in hemoglobin becomes more protonated and protein shifts to T-state. O2 binding affinity decreases. CO2 binding in Hemoglobin - SOLUTION Forms carbonic acid that shifts hemoglobin to T-state. O2 binding affinity decreases. Used in the peripheral tissues. BPG (2,3-bisphosphoglycerate) - SOLUTION Greatly reduces hemoglobin's affinity for O2 by binding allosterically. Stabilizes T-state. Transfer of O2 can improve because increased delivery in tissues can outweigh decreased binding in the lungs. Michaelis-Menton Equation - SOLUTION V0 = (Vmax[S]) / (Km + [S]) Km in Michaelis-Menton - SOLUTION Km = [S] when V0 = 0.5(Vmax) Michaelis-Menton Graph - SOLUTION
Furanose is a 5-membered ring. Mutarotation - SOLUTION Conversion from α to ß forms of the sugar at the anomeric carbon. Anomeric Carbon - SOLUTION Carbon that is cyclized. Always the same as the aldo or keto carbon in the linear form. α vs. ß sugars - SOLUTION α form has -OR/OH group opposite from the - CH2OH group. ß form has -OR/OH group on the same side as the -CH2OH group. Starch - SOLUTION Found in plants. D-glucose polysaccharide. "Amylose chain". Unbranched. Has reducing and non-reducing end. Amylose Chain - SOLUTION Has α-1,4-linkages that produce a coiled helix similar to an α-helix. Has a reducing and non-reducing end. Amylopectin - SOLUTION Has α-1,4-linkages. Has periodic α-1,6-linkages that cause branching. Branched every 24-30 residues. Has reducing and non-reducing end. Reducing Sugar - SOLUTION Free aldehydes can reduce FeIII or CuIII. Aldehyde end is the "reducing" end. Glycogen - SOLUTION Found in animals. Branched every 8-12 residues and compact. Used as storage of saccharides in animals. Cellulose - SOLUTION Comes from plants. Poly D-glucose. Formed from ß-1,4-linkage. Form sheets due to equatorial -OH groups that H-bond with other chains. Chitin - SOLUTION Homopolymer of N-acetyl-ß-D-glucosamine. Have ß- 1,4-linkages. Found in lobsters, squid beaks, beetle shells, etc. Glycoproteins - SOLUTION Carbohydrates attached to a protein. Common outside of the cell. Attached at Ser, Thr, or Asn residues. Membrane Translayer Flip-Flop - SOLUTION Typically slow, but can be sped up with Flippase, Floppase, or Scramblase.
Membrance Fluidity - SOLUTION Membrane must be fluid. Cis fats increase fluidity, trans fats decrease fluidity. Type I Integral Membrane Protein - SOLUTION Membrane protein with C- terminus inside and N-terminus outside Type II Integral Membrane Protein - SOLUTION Membrane protein with N- terminus inside and C-terminus outside Type III Integral Membrane Protein - SOLUTION Membrane protein that contains connected protein helices Type IV Integral Membrane Protein - SOLUTION Membrane protein that contains unconnected protein helices Bacteriorhodopsin - SOLUTION Type III integral membrane protein with 7 connected helices. ß-Barrel Membrane Protein - SOLUTION Can act as a large door. Whole proteins can fit inside. α-hemolysin - SOLUTION Secreted as a monomer. Assembles to punch holes in membranes. Cardiolipin - SOLUTION "Lipid staple" that ties two proteins (or complexes) together in a membrane. Formed from two phosphoglycerols. Hydrolysis of Nucleotides - SOLUTION Base hydrolyzes RNA, but not DNA. DNA is stable in base because of 2' deoxy position. Chargaff's Rule - SOLUTION Ratio of A:T and G:C are always equal or close to 1 DNA Double-Helix - SOLUTION Opposite strand direction. 3.4Å distance between complementary bases. 36Å for one complete turn. A-form DNA - SOLUTION Condensed form of DNA. Deeper major groove and shallower minor groove.
Step 5 of Epinephrine Signal Transduction - SOLUTION cAMP phosphorylates PKA, activating it Step 6 of Epinephrine Signal Transduction - SOLUTION Phosphorylated PKA causes an enzyme cascade causing response to epinephrine Step 7 of Epinephrine Signal Transduction - SOLUTION cAMP is degraded, reversing activation of PKA. α-subunit hydrolyzes GTP to GDP and becomes inactivated. cAMP - SOLUTION Secondary messenger in GPCR signalling. Formed from ATP by adenylyl cyclase. Activates PKA (protein kinase A). AKAP - SOLUTION Anchoring protein that binds to PKA, GPCR, and adenylyl cyclase. GAPs (GTPase activator proteins) - SOLUTION Increase activity of GTPase activity in α-subunit of GPCR. ßARK and ßarr - SOLUTION Used in desensitization. ßARK phosphorylates receptors and ßarr draws receptor into the cell via endocytosis RTKs (Receptor Tyrosine Kinases) - SOLUTION Have tyrosine kinase activity that phosphorylates a tyrosine residue in target proteins INSR (Insulin Receptor Protein) - SOLUTION Form of RTK. Catalytic domains undergo auto-phosphorylation. INSR signalling cascade - SOLUTION INSR phosphorlates IRS-1 that causes a kinase cascade. INSR cross-talk - SOLUTION INSR causes a kinase cascade that alters gene expression and phosphorlates ß-adrenergic receptor causing its endocytosis. NADH - SOLUTION FADH2 - SOLUTION Single-electron transfer
NADPH - SOLUTION
FMN - SOLUTION Single electron transfer. Step 1 of Glycolysis - SOLUTION Glucose --> Glucose 6-phosphate. Uses hexokinase enzyme. ATP --> ADP Step 2 of Glycolysis - SOLUTION Glucose 6-phosphate <--> Fructose 6- phosphate Uses phosphohexose isomerase enzyme. Step 3 of Glycolysis - SOLUTION Fructose 6-phosphate --> Fructose 1,6- bisphosphate Uses PFK-1 (phosphofructokinase-1) enzyme. ATP --> ADP First Committed Step of Glycolysis - SOLUTION Step 3 of Glycolysis. Fructose 6-Phosphate --> Fructose 1,6-bisphosphate. (PFK-1) Step 4 of Glycolysis - SOLUTION Fructose 1,6-bisphosphate <--> dihydroxyacetone + glyceraldehyde 3-phosphate. Uses aldolase enzyme. Step 5 of Glycolysis - SOLUTION Dihydroxyacetonephosphate <--> glyceraldehyde 3-phosphate Uses triose phosphate isomerase enzyme. Step 6 of Glycolysis - SOLUTION Glyceraldehyde 3-Phosphate + Pi <--> 1,3-biphosphoglycerate. Uses G3P dehydrogenase enzyme. NAD+ <--> NADH First Energy Yielding Step of Glycolysis - SOLUTION Step 6 of Glycolysis. G3P + Pi <--> 1,3-bisphosphoglycerate Step 7 of Glycolysis - SOLUTION 1,3-bisphosphoglycerate + ADP <--> 3- phosphoglycerate + ATP Uses phosphoglycerate kinase enzyme.
TPP Cofactor - SOLUTION Common acetaldehyde carrier. Used in pyruvate decarboxylase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase Bypass Reactions in Gluconeogenesis - SOLUTION Steps 1,3, and 10 must be bypassed. Gluconeogenic Bypass of Step 10 - SOLUTION Bicarbonate + Pyruvate --> Oxaloacetate Pyruvate decarboxylate (biotin) ATP --> ADP Oxaloacetate --> PEP PEP carboxykinase GTP --> GDP + CO Gluconeogenic Bypass of Step 3 - SOLUTION Fructose 1,6-bisphosphate
- H2O --> Fructose 6-phosphate + Pi Uses FBPase-1 (coordinated with PFK-1) Gluconeogenic Bypass of Step 1 - SOLUTION Glucose 6-phosphate + H2O --> Glucose + Pi Uses glucose 6-phosphatase. Cost of Gluconeogenesis - SOLUTION 4 ATP, 2 GTP, and 2 NADH Oxidative Pentose Phosphate Pathway - SOLUTION Uses glucose 6- phosphate to produce 2 NADPH and ribose 5-phosphate used for biosynthesis Non-Oxidative Pentose Phosphate Pathway - SOLUTION Regenerates glucose 6-phosphate from ribose 5-phosphate. Uses transketolase and transaldolase enzymes. Transketolase - SOLUTION Transfers a two-carbon keto group Transaldolase - SOLUTION Transfers a three-carbon aldo group Enzyme Km and Substrate Concentration - SOLUTION Most enzymes have a Km that is near the concentration of the substrate.
Fructose 2,6-bisphosphate - SOLUTION Not a glycolytic intermediate. Interconverts between fructose 2,6-bisphosphate and fructose 6-phosphate using PFK-2 and FBPase- Regulation with fructose 2,6-bisphosphate - SOLUTION Activates PFK- encouraging glycolysis. Inhibits FBPase-1 discouraging gluconeogenesis Regulation of Pyruvate Kinase - SOLUTION Inhibited by ATP, Acetyl-Coa, Alanine, long-chain FA's. PDH (Pyruvate Dehydrogenase Complex) - SOLUTION Large complex that converts pyruvate + Coa --> Acetyl-Coa + CO Uses pyruvate dehydrogenase, dihydolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Inhibited by phosphorylation by ATP. Pyruvate Dehydrogenase - SOLUTION E1 domain of the PDH complex. Contains TPP cofactor. Releases CO2. Dihydrolipoyl Transacetylase - SOLUTION E2 domain of the PDH complex. Catalyzes formation of Acetyl-CoA. Has oxidized, acyl, and reduced lipoyllysine. Dihydrolipyl Dehydrogenase - SOLUTION E3 domain of the PDH complex. Catalyzes regeneration of the lipoyllysine using FAD --> FADH Step 1 of the Citric Acid Cycle - SOLUTION Acetyl-CoA + Oxaloacetate --> Citrate Uses citrate synthase enzyme H2O --> CoA Rate-limiting Step of the Citric Acid Cycle - SOLUTION Step 1 Acetyl-Coa + Oxaloacetate --> Citrate Step 2 of the Citric Acid Cycle - SOLUTION Citrate <--> Isocitrate Uses aconitase enzyme H2O <--> H2O Step 3 of the Citric Acid Cycle - SOLUTION Isocitrate --> α-ketoglutarate
CO2 Producing Steps of the Citric Acid Cycle - SOLUTION Steps 3 and 4 Isocitrate --> α-ketoglutarate α-ketoglutarate --> Succinyl-CoA Biotin Structure - SOLUTION Biotin Function - SOLUTION Prosthetic group that serves as a CO2 carrier to separate active sites on an enzyme Regulation of the Citric Acid Cycle - SOLUTION Regulation occurs at Steps 1, 2, 4, and 5. High energy molecules (ATP, Acetyl-CoA, NADH) inhibit while low-energy molecules (ADP, AMP, CoA, NAD+) activate these steps Glyoxylate Cycle - SOLUTION Found in plants. Produces succinate from 2 acetyl-CoA. Allows oxaloacetate in the CAC to be used in gluconeogenesis. Uses 3 steps from the CAC. Different Steps in the Glyoxylate Cycle - SOLUTION Isocitrate --> Glyoxylate (+ succinate) Uses isocitrate lyase Glyoxylate (+ acetyl-coA) --> Malate Uses malate synthase Step 1 of ß-oxidation - SOLUTION Fatty acyl-CoA --> trans-Δ2-enoyl-CoA Uses acyl-CoA dehydrogenase FAD --> FADH Results in trans double-bond Step 2 of ß-oxidation - SOLUTION trans-Δ2-enoyl-CoA (+ H2O) --> L-ß- hydroxy-acyl-CoA Uses enoyl-CoA hydratase TFP (Trifunctional Protein) - SOLUTION Protein complex that catalyzes the last three reactions of ß-oxidation. Hetero-octamer (α4ß4)
Step 3 of ß-oxidation - SOLUTION L-ß-hydroxy-acyl-CoA --> ß-ketoacyl- CoA Uses ß-ketoactyl-CoA dehydrogenase NAD+ --> NAD+ Oxidation of Odd-numbered FA's - SOLUTION Results in propionyl-CoA formation. Propionyl-CoA can be converted to succinyl-CoA and used in the CAC Step 4 of ß-oxidation - SOLUTION ß-ketoacyl-CoA (+ CoA) --> Fatty acyl- Coa (shorter) Uses thiolase enzyme ß-oxidation in plants - SOLUTION Electrons are passed directly to molecular oxygen releasing heat and H2O2 instead of the respiratory chain. ω-oxidation - SOLUTION Similar to ß-oxidation but occurs simultaneously on both ends of the molecule. α-oxidation - SOLUTION Form of oxidation of branched FA's. Produced propionyl-CoA that must be converted to succinyl-CoA for use in the CAC Ketone bodies - SOLUTION Consists of Acetoacetate, Acetone, and D-ß- hydroxybutryate. Formation begins from condensation of 2 acetyl-CoA --> Acetoacetyl-CoA (+ CoA) D-ß-hydroxybutryate can be broken into 2 acetyl-CoA and used as fuel. Zymogen - SOLUTION An inactive precursor of an enzyme, activated by various methods (acid hydrolysis, cleavage by another enzyme, etc.) PLP Structure - SOLUTION Amidotransferase - SOLUTION Uses a PLP group to transfer amino group from an amino acid to α-ketoglutarate to form L-glutamate and an α- ketoglutarate.
pKa of Arginine R-group - SOLUTION 12. pKa of Aspartate R-group - SOLUTION 3. pKa of Cysteine R-group - SOLUTION 8 pKa of Glutamate R-group - SOLUTION 4 pKa of Histidine R-group - SOLUTION 6. pKa of Lysine R-group - SOLUTION 10. pKa of Tyrosne R-group - SOLUTION 10 FAD Structure - SOLUTION Q (Ubiquinone/Coenzyme Q) Structure - SOLUTION Q (Ubiquinone/Coenzyme Q) Function - SOLUTION Lipid soluble electron carrier. Carries 2 electrons with 2 H+. ETC (Electron Transport Chain) - SOLUTION Consists of 4 functional protein complexes. Complex I in the ETC - SOLUTION Accepts two electrons from NADH via an FMN cofactor. Transfers 4H+ to Pside and 2H+ to Q Complex II in the ETC - SOLUTION Succinate dehydrogenase. Accepts two electrons electrons from succinate via an FAD group. Q --> QH Complex III in the ETC - SOLUTION Transfers two electrons from QH2 to cytochrome c via the Q-cycle. Transfers 4H+ to Pside. Complex IV in the ETC - SOLUTION Transfers electrons from cytochrome c to O2. Four electrons are used to reduce on O2 into two H2O molecules. Transfers 4H+ to Pside Mitochondrial ATP Synthase - SOLUTION Consists of F1 and F0 domains
F1 Domain of Mitochondrial ATP Synthase - SOLUTION Hexamer of 3 αß dimers. Catalyze ADP + Pi --> ATP via binding-change model F0 Domain of Mitochondrial ATP Synthase - SOLUTION Causes rotation of γ-subunit via a half channel and H+ gradient Malate-Aspartate Shuttle - SOLUTION Used to maintain gradient of NADH inside of the mitochondria. Involves transport of malate or aspartate; aspartate aminotransferase; and malate dehydrogenase. RuBisCo (Ribulose 1,5-bisphosphate carboxylase/oxygenase) - SOLUTION Incorporates CO2 into ribulose 1,5-bisphosphate and cleaves the 6C intermediate into 2 3-phosphoglycerate. Stage 1 of the Calvin Cycle - SOLUTION 3 ribulose 1-5-bisphosphate + 3 CO2 --> 6 3-phosphoglycerate. Catalyzed by rubisco Mg2+ in Rubisco - SOLUTION Stabilizes negative charge in intermediate and held by Glu, Asp, and carbamoylated Lysine residue Rubisco Activase - SOLUTION Triggers removal of ribulose 1,5- bisphosphate or 2-carboxyaarabinitol 1-phosphate so Lys can be carbamoylated. 2-carboxyarabinitol 1-phosphate - SOLUTION inhibits carbamoylated rubisco. Synthesized in the dark and is broken down by rubisco activase or light. Stage 2 of the Calvin Cycle - SOLUTION 3-phosphoglycerate --> glyceraldehyde 3-phosphate Requires ATP and NADPH Goes through 1,3-bisphosphoglycerate intermediate Stage 3 of the Calvin Cycle - SOLUTION Glyceraldehyde 3-phosphate --> Ribulose 1,5-bisphosphate Requires 3 ATP and uses transketolase (TPP). Only uses 8 of the 9 G3P's produced. One G3P is used to make starch/sucrose.
