11/06/2008Biochemistry: Metabolism I General Metabolism I Andy Howard Introductory Biochemistry 6 November 2008

11/06/2008Biochemistry: Metabolism I Page 2 of 34 What we’ll discuss Metabolism Definitions Pathways Control Feedback Phosphorylation Thermodynamics Kinetics Cofactors Tightly-bound metal ions as cofactors Activator ions as cofactors Cosubstrates Prosthetic groups

11/06/2008Biochemistry: Metabolism I Page 3 of 34 Metabolism Almost ready to start the specifics (chapter 18) Define it! Metabolism is the network of chemical reactions that occur in biological systems, including the ways in which they are controlled. So it covers most of what we do here!

11/06/2008Biochemistry: Metabolism I Page 4 of 34 Intermediary Metabolism Metabolism involving small molecules Describing it this way is a matter of perspective: Do the small molecules exist to give the proteins something to do, or do the proteins exist to get the metabolites interconverted?

11/06/2008Biochemistry: Metabolism I Page 5 of 34 Anabolism and catabolism Anabolism: synthesis of complex molecules from simpler ones Generally energy-requiring Involved in making small molecules and macromolecules Catabolism:degradation of large molecules into simpler ones Generally energy-yielding All the sources had to come from somewhere

11/06/2008Biochemistry: Metabolism I Page 6 of 34 Common metabolic themes Maintenance of internal concentrations of ions, metabolites, enzymes Extraction of energy from external sources Pathways specified genetically Organisms & cells interact with their environment Constant degradation & synthesis of metabolites and macromolecules to produce steady state

11/06/2008Biochemistry: Metabolism I Page 7 of 34 Metabolism and energy

11/06/2008Biochemistry: Metabolism I Page 8 of 34 Pathway A sequence of reactions such that the product of one is the substrate for the next Similar to an organic synthesis scheme (but with better yields!) May be: Unbranched Branched Circular

11/06/2008Biochemistry: Metabolism I Page 9 of 34 Why multistep pathways? Limited reaction specificity of enzymes Control of energy input and output: Break big inputs into ATP-sized inputs Break energy output into pieces that can be readily used elsewhere

11/06/2008Biochemistry: Metabolism I Page 10 of 34 Regulation Organisms respond to change Fastest: small ions move in msec Metabolites: sec Enzymes: minutes to days Flow of metabolites is flux: steady state is like a leaky bucket Addition of new material replaces the material that leaks out the bottom

11/06/2008Biochemistry: Metabolism I Page 11 of 34 Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki

11/06/2008Biochemistry: Metabolism I Page 12 of 34 Feedback and Feed-forward Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway

11/06/2008Biochemistry: Metabolism I Page 13 of 34 Feedback realities Control usually exerted at first committed step (i.e., the first reaction that is unique to the pathway) Controlling element is usually the last element in the path

11/06/2008Biochemistry: Metabolism I Page 14 of 34 Feed-forward Early metabolite activates a reaction farther down the pathway Has the potential for instabilities, just as in electrical feed-forward Usually modulated by feedback

11/06/2008Biochemistry: Metabolism I Page 15 of 34 Activation and inactivation by post-translational modification Most common: covalent phosphorylation of protein usually S, T, Y, sometimes H Kinases add phosphate Protein-OH + ATP  Protein-O-P + ADP … ATP is source of energy and P i Phosphatases hydrolyze phosphoester: Protein-O-P +H 2 O  Protein-OH + P i … no external energy source required

11/06/2008Biochemistry: Metabolism I Page 16 of 34 Phosphorylation’s effects Phosphorylation of an enzyme can either activate it or deactivate it Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated Example: glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme

11/06/2008Biochemistry: Metabolism I Page 17 of 34 Glycogen phosphorylase Reaction: extracts 1 glucose unit from non-reducing end of glycogen & phosphorylates it: (glycogen) n + P i  (glycogen) n-1 + glucose-1-P Activated by phosphorylation via phosphorylase kinase Deactivated by dephosphorylation by phosphorylase phosphatase

11/06/2008Biochemistry: Metabolism I Page 18 of 34 Amplification Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins Thus a single activation event at the kinase level can trigger many events at the target level

11/06/2008Biochemistry: Metabolism I Page 19 of 34 Other PTMs (p. 505) Are there other reversible PTMs that regulate enzyme activity? Yes: Adenylation of Y ADP-ribosylation of R Uridylylation of Y Oxidation of cysteine pairs to cystine

11/06/2008Biochemistry: Metabolism I Page 20 of 34 Evolution of Pathways: How have new pathways evolved? Add a step to an existing pathway Evolve a branch on an existing pathway Backward evolution Duplication of existing pathway to create related reactions Reversing an entire pathway

11/06/2008Biochemistry: Metabolism I Page 21 of 34 Adding a step A  B  C  D  E  P When the organism makes lots of E, there’s good reason to evolve an enzyme E 5 to make P from E. This is how asn and gln pathways (from asp & glu) work E1E1 E2E2 E3E3 E4E4 E5E5 Original pathway

11/06/2008Biochemistry: Metabolism I Page 22 of 34 Evolving a branch Original pathway: D A  B  C X Fully evolved pathway: D A  B  C X E1E1 E2E2 E3E3 E 3a E 3b

11/06/2008Biochemistry: Metabolism I Page 23 of 34 Backward evolution Original system has lots of E  P E gets depleted over time; need to make it from D, so we evolve enzyme E4 to do that. Then D gets depleted; need to make it from C, so we evolve E3 to do that And so on

11/06/2008Biochemistry: Metabolism I Page 24 of 34 Duplicated pathways Homologous enzymes catalyze related reactions; this is how trp and his biosynthesis enzymes seem to have evolved Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination)

11/06/2008Biochemistry: Metabolism I Page 25 of 34 Reversing a pathway We’d like to think that lots of pathways are fully reversible Usually at least one step in any pathway is irreversible (  G o ’ < -15 kJ mol -1 ) Say C  D is irreversible so E 3 only works in the forward direction Then D + ATP  C + ADP + P i allows us to reverse that one step with help The other steps can be in common This is how glycolysis evolved from gluconeogenesis

11/06/2008Biochemistry: Metabolism I Page 26 of 34 Many cofactors are derived from vitamins We justify lumping these two topics together because many cofactors are vitamins or are metabolites of vitamins.

11/06/2008Biochemistry: Metabolism I Page 27 of 34 Family tree of cofactors Cofactors, coenzymes, essential ions, cosubstrates, prosthetic groups: Cofactors (apoenzyme + cofactor  holoenzyme) Essential ions Coenzymes Activator ions (loosely bound) Ions in metalloenzymes Prosthetic groups (tightly bound) Cosubstrates (loosely bound)

11/06/2008Biochemistry: Metabolism I Page 28 of 34 Metal-activated enzymes Absolute requirements for mobile ions Often require K +, Ca 2+, Mg 2+ Example: Kinases: Mg-ATP complex Metalloenzymes: firmly bound metal ions in active site Usually divalent or more Sometimes 1e - redox changes in metal

11/06/2008Biochemistry: Metabolism I Page 29 of 34 Coenzymes Organic moeities that enable enzymes to perform their function: they supply functionalities not available from amino acid side chains Cosubstrates Enter reaction, get altered, leave Repeated recycling within cell or organelle Prosthetic groups Remain bound to enzyme throughout Change during one phase of reaction, eventually get restored to starting state

11/06/2008Biochemistry: Metabolism I Page 30 of 34 Major cosubstrates Facilitate group transfers, mostly small groups Oxidation-reduction participants CosubstrateSourceFunction ATPTransfer P,Nucleotide S-adenosylMetMethyl transfer UDP-glucoseGlycosyl transfer NAD,NADPNiacin2-electron redox Coenzyme APantothenateAcyl transfer TetrahydrofolateFolate1Carbon transfer UbiquinoneLipid-soluble e - carrier

11/06/2008Biochemistry: Metabolism I Page 31 of 34 Major prosthetic groups Transfer of larger groups One- or two-electron redox changes Prosth.gp.SourceFunction FMN, FADRiboflavin1e - and 2e - redox transfers TPPThiamine2-Carbon transfers with C=O PLPPyridoxineAmino acid group transfers BiotinBiotinCarboxylation, COO - transfer Adenosyl-CobalaminIntramolec. rearrangements cobalamin MeCobal.CobalaminMethyl-group transfers LipoamideTransfer from TPP RetinalVitamin AVision Vitamin KVitamin KCarboxylation of glu residues

11/06/2008Biochemistry: Metabolism I Page 32 of 34 Adenosine triphosphate Synthesizable in liver (chapter 18) Building block for RNA Participates in phosphoryl-group transfer in kinases Source of other coenzymes

11/06/2008Biochemistry: Metabolism I Page 33 of 34 S-adenosylmethionine Made from methionine and adenosine Sulfonium group is highly reactive: can donate methyl groups Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University

11/06/2008Biochemistry: Metabolism I Page 34 of 34 UDP-glucose Most common donor of glucose Formed via: Glucose-1P + UTP  UDP-glucose + PP i Reaction driven to right by PP i hydrolysis Structure courtesy of UIC Pharmacy Program