Lecture 13 (10/13/17)

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1 Lecture 13 (10/13/17) Reading: Ch6; , Problems: Ch4 (text); 2, 3 NXT (after xam 2) Reading: Ch6; , , Problems: Ch6 (text); 5, 6, 7, 24 OUTLIN NZYMS: Binding & Catalysis A. Binding 1. Binding curves; How tight? a. Hyperbolic saturation b. Sigmoidal cooperativity B. Catalysis 1. Catalytic power a. Proficiency b. assay of rate c. rate versus [] C. Nomenclature 1. nzyme helpers: Cofactors 2. Reaction Nomenclature (kinetic mechanism) 3. nzyme Nomenclature (names) a. Trivial b. nzyme Commission (C#) Lecture 13 (10/13/17) 1

2 Binding affinities: examples Binding of receptor and ligand Receptors can be soluble or membrane-bound Similar to binding of enzyme and substrate How do we measure the degree of the affinity? Receptor-Ligand binding R + L k 1 k -1 R L Association reaction R L k 1 k -1 R + L K D = [R][L] [R L] Dissociation reaction What are the units of a dissociation constant? M How about an association constant? M -1 How can we express how TIGHT a ligand binds to a protein? How can we measure how TIGHT a ligand binds to a protein? 16 2

3 : Receptor-Ligand binding Define fraction of R bound to a ligand = Y Y =[R L] / ([R]+[R L]) Use K eq to calculate expression for free R Substitute for free R K D = [R][L] [R L] [R] = K D [R L] / [L] Y =[R L] / (K D [R L] / [L]+[R L]) [R L] / [R L] R T = R + R L [ R L] Y = [ R] T Y =1/(K D /[L] + 1) [L] / [L] Y = [L] K D +[L] quation for hyperbola à y = x/(b+x) Graph [L] vs fraction of receptor that is bound (Y) 17 : Receptor-Ligand binding [ L] Y = hyperbola à y = x/(b+x) KD + [ L] Fraction bound (B) (Y) = [R L] [R] T Hyperbolic Curve (Y) NOT: what is Y when [L] = K d? 3

4 : Receptor-Ligand binding Cooperative Binding: Multiple binding sites R L k 1 R + nl n k -1 Y = K [ L] [ L] D + n (Y) 1.0 Fraction Bound 0.5 n Sigmoidal Curve D Ligand concentration [L] Better/easier binding Difficult/poor binding Lag phase Binding of a ligand at one site can affect the binding at other sites. 19 Substrate (S) Binding nzyme () Product (P) nzyme Substrate Complex (S) nzyme Product Complex (P) Chemistry Four introductory aspects: Now that we have some concept of binding, the first important part of the catalytic cycle, lets discuss the second part of the cycle: Catalysis 1) Rate enhancement 2) How do you measure catalysis (enzyme activity) 3) What is the relationship between reaction rate and []? 4) nzyme nomenclature a.reaction b.helpers c. enzymes 4

5 Catalysis Rate nhancement by This ratio is sometimes called Proficiency (exact definition later) TABL 6-5 Some Rate nhancements Produced by Cyclophilin 10 5 Carbonic anhydrase 10 7 Triose phosphate isomerase 10 9 Carboxypeptidase A Phosphoglucomutase Succinyl-CoA transferase Urease Orotidine monophosphate decarboxylase Uroporphyrinogen decarboxylase Catalysis How do you measure enzyme activity? S P For this, you need an ASSAY. You can either measure the disappearance of S or the appearance of P (or couple to faster reactions that measure these indirectly; called a coupled assay) For example: LDH for pyruvate D-lactate You can measure the loss of NADH or the appearance of NAD + Rate (v) = d[p]/dt Rate (v) = -d[s]/dt Concentration (M) [P] Actually. [S] slopes = rate Time (t) 5

6 Catalysis What is the relationship between reaction rate and []? Concentration (M) [S] More Concentration (M) [S] More Concentration (M) [S] Time (t) Time (t) Time (t) Rate (v) = k cat [] (M sec -1 ) (sec -1 ) (M) The turnover number (k cat ) is the number of catalytic cycles/unit time per enzyme active site. Range of values from <1 to 10 7 sec -1 Rate (v) [] slope = 1.0 No enzyme, no rate Double the [enzyme], double the rate Said to be Diffusion Controlled nzyme nomenclature 1.Reaction Nomenclature 2.nzyme helpers (cofactors) 3.Naming enzymes 6

7 Reaction Nomenclature Substrate (S) Binding nzyme () nzyme Substrate Complex (S) nzyme Product Complex (P) Chemistry S + S S P + P P Product (P) (S P) - Reaction coordinate written as a horizontal line - Substrates binding with arrow down; substrates are denoted in order A, B, C - Products released with arrow up; products are denoted in order P, Q, R - nzyme forms are denoted in order as, F, G - Define the number of substrates and products separately as uni=1, bi=2, ter=3 - Define the order of binding and release separately as ordered or random - Define the relationship of substrates to products as sequential or ping-pong A P What about more This is a uni-uni reaction. (A P) complicated reactions? No need to designate last two when there is only one substrate and one product Reaction Nomenclature A B P Q (AB PQ) Sequential ordered bi random bi Q P A B P Q (AB PQ) Sequential random bi ordered bi B A 7

8 Reaction Nomenclature Sequential ordered bi ordered bi Sequential ordered bi bi Sequential random bi random bi Sequential random bi bi Ping-pong ordered bi ordered bi Ping-pong bi bi Which is the reaction catalyzed by LDH? nzyme Helpers Cofactors are small molecules that some enzymes require for activity. The two main classes of cofactors are coenzymes (organic molecules derived from vitamins) and metals. Covalently bound coenzymes are called prosthetic groups. An enzyme with its cofactor is a holoenzyme. Without the cofactor, the enzyme is called an apoenzyme. Cofactors Metal ions coenzymes Coenzymes (co-substrate) Prosthetic (covalent) 8

9 Naming *Trivial: Nearly all enzymes end with the suffix of -ase. Generally, the names are of the form substrate or product reaction catalyzed. For example, lactate dehydrogenase is for an enzyme that removes a hydrogen (plus 2e, i.e., a hydride) from lactate, yielding the carbonyl in pyruvate. There are two ways of naming enzymes; 1) Trivial and 2) Systematic * Not all possible types listed Bullets are those also given as systematic Name -dehydrogenase Reaction Catalyzed redox/hydride transfer lactate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase -oxidase redox/o2 as oxidizer cytochrome oxidase glucose oxidase -oxygenase redox/o2 incorporated cyclooxygenase Ribulose Bisphosphate Carboxylase Oxygenase -hydroxylase redox/-oh incorporated tyrosine hydroxylase phenylalanine hydroxylase -kinase transfer/pi into substrate from ATP hexose kinase protein kinase A -hydrolase hydrolysis with H2O (esterase, deacylase) trypsin phospholipase C -phosphorylase hydrolysis with Pi instead of H2O glycogen phosphorylase b Thymidine phosphorylase -mutase move Pi from one part of molecule to another phosphoglycerate mutase phosphoglucose mutase -isomerase configuration change triosephosphate isomerase phosphogluco isomerase -synthase synthesis fatty acid synthase nitric oxide synthase -synthetase synthesis that requires ATP aminoacyl-trna synthetases acyl-coa synthetase Naming *Trivial: Nearly all enzymes end with the suffix of -ase. Generally, the names are of the form substrate or product reaction catalyzed. For example, lactate dehydrogenase is for an enzyme that removes a hydrogen (plus 2e, i.e., a hydride) from lactate, yielding the carbonyl in pyruvate. There are two ways of naming enzymes; 1) Trivial and 2) Systematic * Not all possible types listed Bullets are those also given as systematic Name -dehydrogenase Reaction Catalyzed redox/hydride transfer lactate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase -oxidase redox/o2 as oxidizer cytochrome oxidase glucose oxidase -oxygenase redox/o2 incorporated cyclooxygenase Ribulose Bisphosphate Carboxylase Oxygenase -hydroxylase redox/-oh incorporated tyrosine hydroxylase phenylalanine hydroxylase -kinase transfer/pi into substrate from ATP hexose kinase protein kinase A -hydrolase hydrolysis with H2O (esterase, deacylase) trypsin phospholipase C -phosphorylase hydrolysis with Pi instead of H2O glycogen phosphorylase b Thymidine phosphorylase -mutase move Pi from one part of molecule to another phosphoglycerate mutase phosphoglucose mutase -isomerase configuration change triosephosphate isomerase phosphogluco isomerase -synthase synthesis fatty acid synthase nitric oxide synthase -synthetase synthesis that requires ATP aminoacyl-trna synthetases acyl-coa synthetase 9

10 Systematic Types Reaction xamples Type Sub-type Sub-class enzyme specific* Great website: C numbers ( -oxidoreductase redox -transferase -hydrolase -lyase -isomerase -ligase Nitrogen-group transfer transfer hydrolysis bond cleavage Intramolecular configuration change configuration change synthesis *this is specific for alcohol dehydrogenase! At alcohol Peptide bonds C-C bond cleavage ster linkage C# Name Trivial Name Uses NAD L-lactate:NADH oxidoreductase lactate dehydrogenase D-glyceraldehyde-3-phosphate:NAD + oxidoreductase glyceraldehyde-3-phosphate dehydrogenase β-d-glucose:oxygen 1-oxidoreductase glucose oxidase L-phenylalanine,tetrahydrobiopterin:oxygen phenylalanine hydroxylase oxidoreductase (4-hydroxylating) Primary amine L-aspartate:2-oxoglutarate aminotransferase Aspartate aminotransferase Acyl-CoA:malonyl-CoA C-acyltransferase fatty acid synthase ATP:D-hexose 6-phosphotransferase hexose kinase Serine mechanism Serine endopeptidylamino acid hydrolase trypsin Phosphatidylcholine:cholinephosphohydrolase phospholipase C Aldehyde product Fructose 1,6-bisphosphate:triosephosphate lyase aldolase (S)-malate hydro-lyase fumarase oxo-acid carboxy-lyase (aldehyde-forming) pyruvate decarboxylase Aldose:ketose Glucose-6-phophate isomerase phosphoglucoisomerase D-glyceraldehyde-3-phosphate:aldose-ketose-isomerase triosephosphate isomerase Proline racemase proline racemase D-phosphoglycerate 2,3-phosphomutase phosphoglycerate mutase (2,3-diphosphoglycerate-dependent) Only 1 subclass Tyrosine aminoacyl-trna ligase Tyr-aminoacyl-tRNA synthetases Pyruvate:carbon-dioxide ligase (ADP-forming) pyruvate carboxylase Systematic Types Reaction xamples Type Sub-type Sub-class enzyme specific* Great website: C numbers ( -oxidoreductase redox -transferase -hydrolase -lyase -isomerase -ligase Nitrogen-group transfer transfer hydrolysis bond cleavage Intramolecular configuration change configuration change synthesis with ATP *this is specific for alcohol dehydrogenase! At alcohol Peptide bonds C-C bond cleavage ster linkage C# Name Trivial Name Uses NAD L-lactate:NADH oxidoreductase lactate dehydrogenase D-glyceraldehyde-3-phosphate:NAD + oxidoreductase glyceraldehyde-3-phosphate dehydrogenase β-d-glucose:oxygen 1-oxidoreductase glucose oxidase L-phenylalanine,tetrahydrobiopterin:oxygen phenylalanine hydroxylase oxidoreductase (4-hydroxylating) Primary amine L-aspartate:2-oxoglutarate aminotransferase Aspartate aminotransferase Acyl-CoA:malonyl-CoA C-acyltransferase fatty acid synthase ATP:D-hexose 6-phosphotransferase hexose kinase Serine mechanism Serine endopeptidylamino acid hydrolase trypsin Phosphatidylcholine:cholinephosphohydrolase phospholipase C Aldehyde product Fructose 1,6-bisphosphate:triosephosphate lyase aldolase (S)-malate hydro-lyase fumarase oxo-acid carboxy-lyase (aldehyde-forming) pyruvate decarboxylase Aldose:ketose Glucose-6-phophate isomerase phosphoglucoisomerase D-glyceraldehyde-3-phosphate:aldose-ketose-isomerase triosephosphate isomerase Proline racemase proline racemase D-phosphoglycerate 2,3-phosphomutase phosphoglycerate mutase (2,3-diphosphoglycerate-dependent) Only 1 subclass Tyrosine aminoacyl-trna ligase Tyr-aminoacyl-tRNA synthetases Pyruvate:carbon-dioxide ligase (ADP-forming) pyruvate carboxylase 10

11 Naming Correlation of trivial and systematic: OTHLIL Naming Oxidoreductase: oxidation-reduction reaction Transferase: transfer of functional group between molecules

12 Naming Hydrolase: hydrolysis (addition of water breaks bond) Lyase: addition of group to a double bond or removal of group to form double bond without hydrolysis or oxidation Naming Isomerase: transfer of functional group within a molecule (rearrangement) Ligase: joining of two molecules (bond formation) coupled with ATP hydrolysis