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

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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 (K m) 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 k cat and K m 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 K d 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; g z 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 B 0||B 1 (“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.

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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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Acknowledgements

The authors would like to thank Krzysztof P. Bzymek for helpful discussions. This work was supported by the National Science Foundation (CHE-0549221, R.C.H.) and the Medical College of Wisconsin Research Affairs Committee (B.B.). The Bruker ESP-300E EPR spectrometer was purchased with funds provided by the National Science Foundation (BIR-9413530) and XSophe was purchased with funds from the National Institutes of Health (NIH RR01008, B.B.).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Utah State University, Logan, UT, 84322-0300, USA

    Ryan Davis, David Bienvenue, Sabina I. Swierczek, Danuta M. Gilner, Lakshman Rajagopal & Richard C. Holz

  2. National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, 53226-0509, USA

    Brian Bennett

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  1. Ryan Davis
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  2. David Bienvenue
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  3. Sabina I. Swierczek
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  7. Richard C. Holz
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Correspondence to Richard C. Holz.

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Davis, R., Bienvenue, D., Swierczek, S.I. et al. Kinetic and spectroscopic characterization of the E134A- and E134D-altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae . J Biol Inorg Chem 11, 206–216 (2006). https://doi.org/10.1007/s00775-005-0071-8

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