Investigations of enzyme mechanisms

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Investigations of enzyme mechanisms
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NMR approaches to mechanistic enzymology

• Investigations of pathways
• Determination of pKa values
• Identification and characterization of binding
  sites
• Identification of (trapped) intermediates
• Determination of regio- and stereospecificity of
  reaction mechanisms
• Face from which protons are abstracted from NADH
• Investigations of hydrogen bonding

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Two examples from recent literature that
illustrate the interplay between NMR and X-ray

• Alkaline phosphatase
• K. M. Holtz and E. R. Kantrowitz, FEBS Lett.,
  462, 7-11 (1999)
• Pancreatic phospholipase
• C. Yuan and M. D. Tsai, Biochim. Biophys. Acta
  1441, 215 (1999)

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E. coli alkaline phosphatase
Double in-line displacement mechanism supported
by X-ray and NMR data Homodimer 449 aa /
monomer Two active sites 30 Å apart Each active
site contains 3 metal binding sites A M1
Zn2 (required) B M2 Zn2 (enhances
activity) C M3 Mg2
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E.Pi Ser102 phosphate
hydrolysis
transphosphorylation
Double displacement with retention of
configuration
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E-P Ser102 phosphate
hydrolysis
31P NMR studies of the hydrolysis reaction E-P
accumulates at acidic pH ? hydrolysis of E-P is
rate limiting 31P inversion transfer rate is pH
independent between pH 6 and 9 ? rate of
dissociation of E.Pi is 30 s-1, close to turnover
rate at low pH (lt 6), dephosphorylation is rate
limiting Zinc-coordinated hydroxide nucleophile
controls the rate-limiting step
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113Cd NMR chemical shift changes when phosphate
acceptors are present
Replacement of Zn2 with Cd2 raises the
sigmoidal pH midpoint of the E-P E.Pi
equilibrium from 5 to 9 or 10
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31P NMR little chemical shift change of E-P
when different metals are substituted
Led to incorrect conclusion that the metal does
not interact with the phosphate in
E-P (corrected by X-ray structure)
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Zinc-coordinated water molecule was observed in
X-ray structure of E-P Both zinc ions have a
direct role in catalysis
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Two in-line displacement steps, each with a
trigonal bipyramidyl transition state Supported
by X-ray structure of vanadate complex
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E.Pi complex
transition state
vanadate complex
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Mutagenesis of Ser120 optimal nucleophile, not
absolutely required Cys works moderately well
(slower by factor of 100) Ala lowers rate by
10,000 but mutant is still 100,000 better than
non-enzymatic reaction (Zn-coordinated water?)
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Arg166 serves to bind phosphate / substrate
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Asp101, Asp153, Lys328 form secondary
interactions to phosphate mutagenesis alters
activity kcat D153G (5-fold higher) faster
phosphate release kcat D101S (35-fold higher)
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Role of octahedral magnesium stabilizes
most-active conformation of the enzyme orients
Asp51 oxygens for role in electron withdrawing /
donating
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Pancreatic phospholipase A2
Member of PLA2 superfamily Hydrolyzes sn-2 ester
bond of phospholipids Water-soluble, works at
water-lipid interface scooting mode catalysis
(stays bound to membrane) hopping mode kinetics
(hops in aqueous phase) Features Ca2
dependence Asp-His diad hydrophobic
channel interfacial binding site hydrogen
binding network
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Pancreatic phospholipase A2
X-ray structures (Drenth / Hol) NMR
structures porcine PLA2 free (Kaptein) porcine
PLA2 bound to micellar interface (Kaptein) bovine
PLA2 free (Tsai)
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Interfacial catalysis represents binding to
interface, which is distinct from substrate
binding E ? E ? ES ? EP ? E P Putative
interface binding site (IBS) ring of cationic and
hydrophobic residues
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