Catalysis by approximation Bringing two substrates together * Nucleoside monophosphate (NMP) kinases * Adenylate kinase, guanylate kinase * Challenge to promote phosphoryl transfer from NTP to NMP without promoting NTP hydrolysis

NMP kinases Conserved NTP binding domain * Central -sheet surrounded by -helices * Loop between first -strand and first helix * Gly-X-X-X-X-Gly-Lys * P-loop, also present in a wide variety of important nucleotide-binding proteins

NMP kinases ATP- Mg2+ complex true substrate * Enhances the binding energy * Reduces non-specific ionic interactions between enzyme and ATP * Hold nucleotide in well-defined conformation

NMP kinases ATP binding induces large conformational changes * Classical example of induced fit * P-loop closes down on top of the ATP chain * Movement of P-loop permits the top domain of the enzyme to move down to form a lid over the bound nucleotide * Binding of second substrate (NMP) induces additional conformational changes bringing the reactive parts of the two substrates close together * Prevents the wasteful transfer of the phosphoryl group to water

Multienzyme catalysis Bringing enzymes together * Structural integration of different kinds of enzymes makes the coordinated catalysis of a complex reaction possible * The proximity of one enzyme to another increases the overall reaction rate and minimizes side reactions * Pyruvate dehydrogenase complex

Pyruvate dehydrogenase complex Member of a family of homologous giant complexes with molecular masses ranging from 4-10 million daltons * -ketoglutarate dehydrogenase * branched chain -ketoacid dehydrogenase * acetoin dehydrogenase

Dihydrolipoyl transacetylase (E2) Core of the complex * 8 catalytic trimers * N-terminal domain contains a bound lipoamide cofactor that acts as a swinging arm * Flexible arm has the ability to call on each active site of E1, E2 and E3 in turn

Multifunctional enzyme complex in eukaryotes Fatty acid synthase Multifunctional enzyme complex in eukaryotes * All enzyme components linked in a large polypeptide chain * Mammalian enzyme: dimer 2x260 kDa * Three domains joined by flexible regions

Mammalian fatty acid synthase 7 different catalytic sites present on a single polypeptide chain * Domain 1: substrate entry and condensation unit * Domain 2: reduction unit * Domain 3: palmitate release unit The arenas of catalytic action are the interfaces between domains on opposite chains

Mammalian fatty acid synthase Flexibility and 20-Å maximal length of the phosphopantetheinyl moiety are critical for the function of the multienzyme complex * No large structural rearrangements needed * Substrate is on a long, flexible arm that can reach each of the numerous active sites * Enhanced efficiency because intermediates are directly transferred from one active site to the next

Multienzyme complexes Many eukaryotic multienzyme complexes are multifunctional proteins in which different enzymes are linked covalently Advantages * Synthetic activity of different enzymes is coordinated * Covalently joined enzymes are more stable than one formed by noncovalent attractions