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Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions

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Abstract

Mammalian ketimine reductase is identical to μ-crystallin (CRYM)—a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones l-thyroxine and 3,5,3′-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-l-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors’ descriptions) (AECK), Δ1-piperideine-2-carboxylate (P2C), Δ1-pyrroline-2-carboxylate (Pyr2C) and Δ2-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

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Abbreviations

AECK:

S-(2-Aminoethyl)-l-cysteine ketimine

CE:

Catalytic efficiency

CRYM:

μ-Crystallin

CTBP:

Cytosolic thyroid hormone binding protein

CysK:

Cystathionine ketmine

DAAO:

d-Amino acid oxidase

DIT:

3,5-Diiodo-l-tyrosine

DTT:

Dithiothreitol

LK:

Lanthionine ketimine

L-PA:

l-Pipecolic acid

P2C:

Δ1-Piperideine-2-carboxylate

OTE:

Oxalyl thioester

Pyr2C:

Δ1-Pyrroline-2-carboxylate

SEM:

Standard error of the mean

T2C:

Δ2-Thiazoline-2-carboxylate

TL2C:

Thiazolidine-2-carboxylate

T2 :

3,5-Diiodothyronine

T3 :

3,5,3′-l-Triiodothyronine

rT3 :

3,3′,5′-l-Triiodothyronine, reverse T3

T4 :

l-Thyroxine

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Acknowledgments

This research was funded by a Macquarie University Research Excellence Scholarship.

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The authors have nothing to declare.

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Authors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia

    André Hallen, Joanne F. Jamie & Peter Karuso

  2. Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595, USA

    Arthur J. L. Cooper

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  1. André Hallen
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Correspondence to André Hallen.

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Hallen, A., Cooper, A.J.L., Jamie, J.F. et al. Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions. Neurochem Res 40, 1252–1266 (2015). https://doi.org/10.1007/s11064-015-1590-5

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