Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L- carnitine and/or R- -lipoic acid

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

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Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09787-y ◽

2019 ◽

Vol 103 (10) ◽

pp. 4053-4064 ◽

Cited By ~ 3

Author(s):

Shouji Takahashi ◽

Kohei Osugi ◽

Yuya Shimekake ◽

Akira Shinbo ◽

Katsumasa Abe ◽

...

Keyword(s):

Binding Affinity ◽

Substrate Binding ◽

Thermophilic Fungus ◽

Thermomyces Dupontii

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Hyaluronan–CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

The Journal of Cell Biology ◽

10.1083/jcb.200202120 ◽

2002 ◽

Vol 158 (6) ◽

pp. 1133-1144 ◽

Cited By ~ 62

Author(s):

Paola Spessotto ◽

Francesca Maria Rossi ◽

Massimo Degan ◽

Raffaele Di Francia ◽

Roberto Perris ◽

...

Keyword(s):

Cell Migration ◽

Bone Resorption ◽

Cell Motility ◽

Binding Affinity ◽

Antisense Oligonucleotides ◽

Substrate Binding ◽

Down Regulation ◽

Multinucleated Cells ◽

Cell Substrate

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell–substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9–specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA–CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.

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Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(01)00272-3 ◽

2001 ◽

Vol 1511 (1) ◽

pp. 123-133 ◽

Cited By ~ 11

Author(s):

Mohsen Lachaal ◽

Robert A Spangler ◽

Chan Y Jung

Keyword(s):

Binding Affinity ◽

Adenosine Triphosphate ◽

Glucose Transporter ◽

Substrate Binding ◽

Glucose Transporter Glut1 ◽

Transporter Glut1

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Lipid oxidation by heart mitochondria from young adult and senescent rats

Biochemical Journal ◽

10.1042/bj1700285 ◽

1978 ◽

Vol 170 (2) ◽

pp. 285-295 ◽

Cited By ~ 91

Author(s):

Richard G. Hansford

Keyword(s):

Age Groups ◽

Heart Mitochondria ◽

Synthetase Activity ◽

Age Group ◽

Carnitine Acetyltransferase ◽

O2 Uptake ◽

First Order ◽

Old Rats ◽

Group 2 ◽

Two Age Groups

1. State-3 (i.e. ADP-stimulated) rates of O2 uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O2 uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[3H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O2 uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O2 uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O2 uptake with palmitoylcarnitine in senescence is discussed.

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