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Catalysis by approximationCatalysis 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
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NMP kinasesNMP 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
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NMP kinasesNMP 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
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NMP kinasesNMP 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
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Multienzyme catalysisMultienzyme 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
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Pyruvate dehydrogenase complexPyruvate 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
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Dihydrolipoyl transacetylase (E2)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
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Fatty acid synthaseFatty 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
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Mammalian fatty acid synthaseMammalian 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
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Mammalian fatty acid synthaseMammalian 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
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Multienzyme complexesMultienzyme 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