Biotinyl-methyl 4-(amidomethyl)benzoate is a competitive inhibitor of human biotinidase

Abstract

Posttranslational modification of histones by biotinylation can be catalyzed by both biotinidase (BTD) and holocarboxylase synthetase. Biotinylation of histones is an important epigenetic mechanism to regulate gene expression, DNA repair, and chromatin remodeling. The role of BTD in histone biotinylation is somewhat ambiguous, given that BTD also catalyzes removal of the biotin tag from histones. Here, we sought to develop BTD inhibitors for future studies of the role of BTD in altering chromatin structure. We adopted an existing colorimetric BTD assay for use in a novel 96-well plate format to permit high-throughput screening of potential inhibitors. Biotin analogs were chemically synthesized and tested for their ability to inhibit human BTD. Seven of these compounds inhibited BTD by 26-80%. Biotinyl-methyl 4-(amidomethyl)benzoate had the largest effect on BTD, causing an 80% inhibition at 1 mM concentration. Enzyme kinetics studies were conducted to determine V(max), K(m) and K(i) for the seven inhibitors; kinetics were consistent with the hypothesis that biotinyl-methyl 4-(amidomethyl)benzoate and the other compounds acted by competitive inhibition of BTD. Finally, biotinyl-methyl 4-(amidomethyl)benzoate did not affect biotin transport in human cells, suggesting specificity in regard to biotin-related processes.

Fig. 1

Standard curve for colorimetric quantification of PABA. Absorbance of derivatives of PABA was measured at 546 nm. The standard deviations are too small to be visible at low amounts of PABA (y = 0.0013x + 0.021; r = 0.998).

Fig. 2

BTD activity depends on the concentration of its substrate, N-(+)-biotinyl-4-amidobenzoic acid. BTD activity was monitored by the release of PABA from N-(+)-biotinyl-PABA. The standard deviations are too small to be visible (y = 0.11x − 1.19; r = 0.989).

Fig. 3

Hydrolysis of N-(+)-biotinyl-4-amidobenzoic acid (60 nmoles/well) depends on the amount of partially purified BTD. The standard deviations are too small to be visible (y = 0.072x − 1.03; r = 0.997).

Fig. 4

Hydrolysis of N-(+)-biotinyl-4-amidobenzoic acid by partially purified BTD is linear for up to 4 hours. Values are means ± SD (n = 6). The standard deviations are too small to be visible.

Fig. 5

Biotinyl 2,4,6-trimethylbenzenamide (1 mM) does not inhibit BTD. The effects of the treatment are not significantly different compared with inhibitor-free controls. The incubation times denote the hours of incubation of partially purified BTD, substrate, and inhibitor. Values are means ± SD (n = 6; P > 0.05).

Fig. 6

Biotinyl-methyl 4-(amidomethyl) benzoate (1 mM) inhibits BTD. Values are means ± SD (n = 6; *P < 0.05 compared with inhibitor-free control). The incubation times denote the hours of incubation of partially purified BTD, substrate, and inhibitor.

Fig. 7

Biotinyl-methyl 4-(amidomethyl) benzoate inhibits BTD activity in a concentration-dependent fashion. Values are means ± SD (n = 6). ^a,b,c,d Bars not sharing the same letter are significantly different from the inhibitor-free control (P < 0.05).

Fig. 8

BTD activity is reduced in a substrate-concentration dependent manner in the presence or absence of 0.5 mM of the inhibitor biotinyl-methyl 4-(amidomethyl) benzoate.

Fig. 9

Lineweaver-Burk plot demonstrating competitive inhibition of BTD by biotinyl methyl 4-(amidomethyl) benzoate. V_max = 0.29 ± 0.009 nmol/min⁻¹/mg⁻¹; K_m = 0.04 ± 0.007 mM; K_i = 0.06 ± 0.01 mM; n = 3.