Kinetic and spectroscopic characterization of the E134A- and E134D-altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae

  • Ryan Davis
  • David Bienvenue
  • Sabina I. Swierczek
  • Danuta M. Gilner
  • Lakshman Rajagopal
  • Brian Bennett
  • Richard C. Holz
Original Paper

Abstract

Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (Km) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine (0%). Examination of the pH dependence of the kinetic constants kcat and Km for E134D-DapE revealed ionizations at pH 6.4, 7.4, and approximately 9.7. Isothermal titration calorimetry experiments revealed a significant weakening in metal Kd values of E134D-DapE. D134 and A134 perturb the second divalent metal binding site significantly more than the first, but both altered enzymes can still bind two divalent metal ions. Structural perturbations of the dinuclear active site of DapE were also examined for two E134-substituted forms, namely E134D-DapE and E134A-DapE, by UV–vis and electron paramagnetic resonance (EPR) spectroscopy. UV–vis spectroscopy of Co(II)-substituted E134D-DapE and E134A-DapE did not reveal any significant changes in the electronic absorption spectra, suggesting that both Co(II) ions in E134D-DapE and E134A-DapE reside in distorted trigonal bipyramidal coordination geometries. EPR spectra of [Co_(E134D-DapE)] and [Co_(E1341A-DapE] are similar to those observed for [CoCo(DapE)] and somewhat similar to the spectrum of [Co(H2O)6]2+ which typically exhibit E/D values of approximately 0.1. Computer simulation returned an axial g-tensor with g(x,y)=2.24 and E/D=0.07; gz was only poorly determined, but was estimated as 2.5–2.6. Upon the addition of a second Co(II) ion to [Co_(E134D-DapE)] and [Co_(E134A-DapE)], a broad axial signal was observed; however, no signals were observed with B0||B1 (“parallel mode”). On the basis of these data, E134 is intrinsically involved in the hydrolysis reaction catalyzed by DapE and likely plays the role of a general acid and base.

Keywords

Hydrolysis Zinc Cobalt Kinetics Electron paramagnetic resonance 

Abbreviations

AAP

Aminopeptidase from Aeromonas proteolytica

CPG2

Carboxypeptidase G2 from Pseudomonas sp. strain RS-16

DapE

dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase)

EcMetAP-I

Methionyl aminopeptidase from Escherichia coli

EPR

Electron paramagnetic resonance

E134

Glutamate-134

E151

Glutamate-151

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

mDAP

meso-Diaminopimelate

d,d-SDAP

N-Succinyl-d,d-diaminopimelate

l,l-SDAP

N-Succinyl-l,l-diaminopimelate

SDAP

N-Succinyl-diaminopimelic acid

Tricine

N-Tris(hydroxymethyl)methylglycine

Tris

Tris(hydroxymethyl)aminomethane

WT

Wild type

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Copyright information

© SBIC 2006

Authors and Affiliations

  • Ryan Davis
    • 1
  • David Bienvenue
    • 1
  • Sabina I. Swierczek
    • 1
  • Danuta M. Gilner
    • 1
  • Lakshman Rajagopal
    • 1
  • Brian Bennett
    • 2
  • Richard C. Holz
    • 1
  1. 1.Department of Chemistry and BiochemistryUtah State UniversityLoganUSA
  2. 2.National Biomedical EPR Center, Department of BiophysicsMedical College of WisconsinMilwaukeeUSA

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