Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

Gaweska, Helena; Pozzi, Michelle Henderson; Schmidt, Dawn M. Z.; McCafferty, Dewey G.; Fitzpatrick, Paul F.

doi:10.1021/bi900499w

Cited by 35 publications

(65 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, there is no evidence for an intermediate in the PAO-catalyzed oxidation of any amine, including those studied here (20, 26). It should be noted that similar small isotope effects have been seen with the flavoprotein amine oxidases LSD1 (38) and tryptophan monooxygenase (21), both of which are in the MAO structural family, and with D-amino acid oxidase (39), which has a different structure. Thus, the combination of steady-state and rapid-reaction kinetics is most consistent with CH bond cleavage being the rate-limiting step in the oxidation of N, N'-dibenzyl-1,4-diaminobutanes by PAO.…”

Section: Discussionmentioning

confidence: 64%

“…The polar nucleophilic mechanism proposed by Edmondson (45) involves concerted formation of a 4a-alkylated isoalloxazine ring and proton abstraction of the substrate α-hydrogen. Direct transfer of a hydride from the α-C of the substrate to the N5 position of the flavin is the consensus mechanism for the D-amino acid oxidase structural family of flavin amine oxidases (16, 46); its applicability to the MAO structural family is supported by kinetic isotope effects and the failure to detect intermediates in the reactions of these enzymes (17–20, 38, 45). The ρ value of −0.59 for PAO at pH 8.6 reported here is most consistent with a direct hydride transfer reaction in which there is little charge development in the transition state for carbon-hydrogen bond cleavage.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Mechanistic Studies of para-Substituted N,N′-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian Polyamine Oxidase

2009

Self Cite

View full text Add to dashboard Cite

The kinetics of oxidation of a series of para-substituted N, N'-dibenzyl-1,4-diaminobutanes by the flavoprotein polyamine oxidase from mouse have been determined to gain insight into the mechanism of amine oxidation by this member of the monoamine oxidase structural family. The k cat /K m values are maximal at pH 9, consistent with the singly charged substrate being the active form. The rate constant for flavin reduction, k red , by N,N'-dibenzyl-1,4-diaminobutane decreases about 5-fold below a pK a of ~8; this is attributed to the need for a neutral nitrogen at the site of oxidation. The k red and k cat values are comparable for each of the N, N'-dibenzyl-1,4-diaminobutanes, consistent with ratelimiting reduction. The deuterium kinetic isotope effects on k red and k cat are identical for each of the N, N'-dibenzyl-1,4-diaminobutanes, consistent with rate-limiting cleavage of the substrate CH bond. The k red values for seven different para-substituted N, N'-dibenzyl-1,4-diaminobutanes correlate with a combination of the van der Waals volume and σ value of the substrates, with ρ values of −0.59 at pH 8.6 and −0.09 at pH 6.6. These results are consistent with direct transfer of a hydride from the neutral CN bond of the substrate to the flavin as the mechanism of polyamine oxidase.The polyamines spermine and spermidine are critical for growth of eukaryotic cells due to the ability of these polycations to bind a variety of macromolecules (1). As a result polyamine metabolism has been a frequent target for the development of anticancer agents (2). Catabolism of spermine and spermidine can occur by two routes (3). In the first pathway to be discovered, spermidine/spermine N1-acetyltransferase converts spermine or spermidine to the N-acetyl form (4). The N1-acetylspermine or N1-acetylspermidine can then be transported out of the cell. Alternatively, the flavoprotein polyamine oxidase (PAO) 1 can catalyze the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively, producing H 2 O 2 (5,6). The more recently discovered flavoprotein spermine oxidase catalyzes the oxidation of spermine directly to spermidine (7,8), bypassing the necessity for acetylation. While PAO is a constitutive enzyme, spermine oxidase is induced by polyamine analogues with antitumor activity (7). The relative importance of the two enzymes in polyamine metabolism in normal and cancer cells remains to be established. PAO belongs to the flavin amine oxidase structural family that includes monoamine oxidase A and B (MAO A/B) (9), lysine-specific demethylase (LSD1) (10), and L-amino acid oxidase *Address correspondence to: Paul F. Fitzpatrick Department of Biochemistry University of Texas Health Science Center at San Antonio 7703 Floyd Curl Dr. San Antonio, TX 78229-3900 ph: 210-567-8264; fax: 210-567-8778 fitzpatrick@biochem.uthscsa.edu. 1 Abbreviations used: PAO, polyamine oxidase; MAO, monoamine oxidase; LSD1, lysine-specific demethylase. NIH Public Access Author ManuscriptBiochemistry. Author manuscrip...

show abstract

Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Mechanistic Studies of para-Substituted N,N′-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian Polyamine Oxidase

2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…We should note that our derived kinetic values are in reasonable agreement with previously reported values with and without CoREST. 13,22,23,30 …”

Section: Resultsmentioning

confidence: 99%

“…30,38 The unmodified 21-mer histone H3 product peptide (H3 1–21 ) was synthesized and purified in a similar fashion. A C-terminal GYG was added to both the substrate and product peptides to allow for quantification of the peptide concentration at 280 nm, using an extinction coefficient of 1490 M −1 cm −1 for tyrosine.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones

et al. 2016

Self Cite

View full text Add to dashboard Cite

Lysine-specific demethylase 1A (KDM1A/LSD1) is a FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively. Although the active site is expanded compared to that of members of the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. We show that full-length histone H3, which lacks any posttranslational modifications, is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM, a value that is approximately 100-fold higher than that of the 21-mer peptide product. The relative H3 affinity is independent of preincubation time, suggesting that H3 rapidly reaches equilibrium with KDM1A. Jump dilution experiments confirmed the increased binding affinity of full-length H3 was at least partially due to a slow off rate (koff) of 1.2 × 10−3 s−1, corresponding to a half-life (t1/2) of 9.63 min, and a residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction, revealing a Kd of 9.02 ± 2.3 nM, a kon of (9.3 ± 1.5) × 104 M−1 s−1, and a koff of (8.4 ± 0.3) × 10−4 s−1. Additionally, no other core histones exhibited inhibition of KDM1A demethylation activity, which is consistent with H3 being the preferred histone substrate of KDM1A versus H2A, H2B, and H4. Together, these data suggest that KDM1A likely contains a histone H3 secondary specificity element on the enzyme surface that contributes significantly to its recognition of substrates and products.

show abstract

Kinetic Isotope Effects in Enzymes

Kohen

Roston

Stojković

et al. 2010

Encyclopedia of Analytical Chemistry

View full text Add to dashboard Cite

Kinetic isotope effects (KIEs) have progressed to become one of the most useful analytical techniques to study enzymatic reactions and can answer a variety of questions ranging from the basic mechanism of a reaction to the structure of a transition state (TS) and the role of quantum mechanical tunneling. Here, we briefly discuss the development of the theory behind KIEs with an emphasis on how it guides the interpretation of experimental data. We then present some of the instrumentation and techniques commonly used for KIE experiments, followed by a number of different examples from the enzymology literature wherein KIEs have been used to probe chemical transformations, with an emphasis on the effects of quantum mechanical tunneling on KIEs.

show abstract

Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

Cited by 35 publications

References 52 publications

Mechanistic Studies of para-Substituted N,N′-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian Polyamine Oxidase

Mechanistic Studies of para-Substituted N,N′-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian Polyamine Oxidase

Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones

Kinetic Isotope Effects in Enzymes

Contact Info

Product

Resources

About