Histidine triad nucleotide‐binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A

Strom, Alexander; Tong, Cher Ling; Wagner, Carston R.

doi:10.1002/1873-3468.13745

Cited by 13 publications

(13 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The final parameter found to be crucial in enabling this RTT based assay was the K D of the P 2 nucleotide product. It has previously been determined that the P 1 side chain product possesses virtually no binding affinity for the HINT1 active site, while the P 2 nucleotide product possesses a relatively poor but observable dissociation constant of ∼100 μM. , Thus, at high substrate concentrations, the resulting high concentration of P 2 can begin to compete with the switch on fluorescent inhibitor after substrate turnover is complete (Figure C). This phenomenon is observed in our experimental fluorescence time course curves, but it does not interfere with the kinetic analysis (Figure B).…”

Section: Resultsmentioning

confidence: 96%

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

2021

Self Cite

View full text Add to dashboard Cite

Enzymatically driven change to the spectroscopic properties of a chemical substrate or product has been a linchpin in the development of continuous enzyme kinetics assays. These assays inherently necessitate substrates or products that naturally comply with the constraints of the spectroscopic technique being used, or they require structural changes to the molecules involved to make them observable. Here we demonstrate a new analytical kinetics approach with enzyme histidine triad nucleotide binding protein 1 (HINT1) that allows us to extract both useful k cat values and a rank-ordered list of substrate specificities without the need to track substrates or products directly. Instead, this is accomplished indirectly using a "switch on" competitive inhibitor that fluoresces maximally only when bound to the HINT1 enzyme active site. Kinetic information is extracted from the duration of the diminished fluorescence when the monitorable inhibitor-bound enzyme is challenged with saturating concentrations of a nonfluorescent substrate. We refer to the loss of fluorescence, while the substrate competes for the fluorescent probe in the active site, as the substrate's residence transit time (RTT). The ability to assess k cat values and substrate specificity by monitoring the RTTs for a set of substrates with a competitive "switch on" inhibitor should be broadly applicable to other enzymatic reactions in which the "switch on" inhibitor has sufficient binding affinity over the enzymatic product.

show abstract

Section: Resultsmentioning

confidence: 96%

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The hHINT continuous fluorescence assay has been utilized for steady-state measurements , as well as for stopped-flow transient kinetic analysis to define the individual rate constants of the hHINT1 reaction coordinate . During the first catalytic step after substrate binding, the active site catalytic nucleophile, H112 is adenylated followed by a rapid release of the tryptamine product.…”

Section: Resultsmentioning

confidence: 99%

“…The hHINT1 construct has a cleavage His tag. hHINT2 with a 36 residue N-terminal truncation (to replicate the cleavage of its mitochondrial localization signal that is removed upon its transport to the mitochondria in vivo) was produced as described. , Briefly, the hHINT2 cDNA was inserted into the pMCSG9 vector with a TEV cleavable N-terminal His-6 tagged maltose binding protein. Rosetta2 (DE3) pLysS competent Escherichia coli were transformed and with the relevant plasmid and cultured in TB media.…”

Section: Methodsmentioning

confidence: 99%

“…Histidine triad nucleotide-binding (HINT) proteins are small, catalytically efficient enzymes known for their nucleotide phosphoramidase and acyl-phosphatase activities, which are critical in the regulation of the μ-opioid (MOR) pathway and chronic pain as well as the management of mitochondrial calcium gradients. − In humans, there are three HINT enzymes, hHINT1, hHINT2, and hHINT3, which have been found to localize in the cytosol, mitochondria, and nucleus, respectively. , hHINT1 bears 61% sequence homology with hHINT2 and previous active site alignments of hHINT1 and hHINT2 crystal structures show that nearly all side-chain residues are identical with a very small root-mean-squared deviation (PDB IDs: 3TW2, 4INI root-mean-square deviation (RMSD) = 0.21Å). , Not surprisingly, hHINT1 and hHINT2 are also known to possess similar substrate specificities ( k cat / K M ) that differ by less than 3-fold for model substrates such as adenosine tryptamine phosphoramidate monoester ( TpAd) (Figure ). …”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides

et al. 2022

Self Cite

View full text Add to dashboard Cite

Human histidine triad nucleotide-binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100), which gate a conserved water channel leading to the active site 13 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state k cat and K m for turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate that K D also changes. hHINT2, like hHINT1, exhibits a burst phase of adenylation, monitored by fluorescent tryptamine release, prior to rate-limiting hydrolysis and nucleotide release. hHINT2 exhibits a much smaller burst-phase amplitude than hHINT1, which is further diminished in hHINT2 R99Q. Kinetic simulations suggest that amplitude variations can be accounted for by a variable fluorescent yield of the E•S complex from changes in the environment of bound TpAd. Isothermal titration calorimetry measurements of inhibitor binding show that these hHINT variants also alter the thermodynamic binding profile. We propose that these altered surface residues engender long-range dynamic changes that affect the orientation of bound ligands, altering the thermodynamic and kinetic characteristics of hHINT active site function. Thus, studies of the cellular roles and proTide activation potential by hHINTs should consider the importance of long-range interactions and possible protein binding surfaces far from the active site.

show abstract

“…The HINT1-Ap 4 A crystal structure revealed the structural basis for the specific chain length for optimal Ap 4 A-HINT interactions, as Ap 3 A chain length was insufficient to interact at nucleotide binding pockets and Ap 5 A binding required a contortion of the phosphodiester linkages (Yu et al, 2019). Recently some doubt has been expressed regarding the ability of HINT1 to bind Ap 4 A, but this could reflect differences in analytical techniques and a lack of post-translational modifications in the HINT1 used (Strom et al, 2020).…”

Section: Ap 4 A-mediated Regulation Of Gene Expression In Eukaryotesmentioning

confidence: 99%

Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger

Ferguson

McLennan

Urbaniak

et al. 2020

Front. Mol. Biosci.

View full text Add to dashboard Cite

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (Np n Ns), the best studied of which is diadenosine tetraphosphate (Ap 4 A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap 4 A and Ap 4 N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the “friend” and “foe” scenarios. The “friend (or alarmone) hypothesis” suggests that Ap n N act as bona fide secondary messengers mediating responses to stress. In contrast, the “foe” hypothesis proposes that Ap n N and other Np n N are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for Ap n N in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to Ap n N. Finally, areas for future research are discussed with possible but unproven roles for intracellular Ap n N to encourage further research into the signaling networks that are regulated by these nucleotides.

show abstract

Histidine triad nucleotide‐binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A

Cited by 13 publications

References 31 publications

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides

Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger

Contact Info

Product

Resources

About