High-Efficiency Incorporation in Vivo of Tyrosine Analogues with Altered Hydroxyl Acidity in Place of the Catalytic Tyrosine-14 of Δ⁵-3-Ketosteroid Isomerase of Comamonas (Pseudomonas) testosteroni:  Effects of the Modifications on Isomerase Kinetics

Abstract

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…As already discussed, Ring et al 66,67 probed the role of catalytic Tyr in the active center of β-galactosidase by the global replacement of all Tyr-side chains with (3-F)Tyr, whereas Brooks and co-workers used (2-F)Tyr to dissect the catalytic mechanism of ketosteroid isomerase. 68 More recently, interesting spectral and dynamic properties of green fluorescent protein upon global substitution of its Tyr-residues with (2-F)Tyr and (3-F)Tyr were reported as well (vide infra). 95,96 Similarly, the appearance of "fluorophenylfingers" discussed above represents a non-invasive fluorinated optical probe designed to introduce dramatic changes in the spectral properties of the protein without compromising its stability and/or biological activity.…”

“…However, anxA5[(3-F)-Tyr] is approximately threefold less active than anxA5 whereas electron transfer processes in azurin seemed not to be altered upon fluorination. Examples of other groups in which fluorination of tyrosine alters enzyme activity include β-galactosidase (fluorination was used to probe whether Tyr is part of the catalytically active portion of the protein), 66,67 ketosteroid isomerase from Pseudomonas testosterone 68 and rat glutathione transferase M1-1. 69 These fluorinated proteins are interesting because of their altered pK a .…”

Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy

“…Differences in the fluorescence emission maxima and excitation maxima were noticed for the red-shifted EYFPs. These differences might results from a partial deprotonation of Tyr203, (2-F)-Tyr203 and (3-F)-Tyr203 at the pH-value of our study, which is above the pK A of these amino acids [22]. Such altered π-π interactions might result in a weakened red-shift of the spectra at pH 10 compared to the values at pH 7.…”

“…Interestingly, the pH-dependence of its absorbance spectra deviated from the other fluorinated proteins resulting in a pK A close to the parent protein [8]. This is noteworthy as the acidity of tyrosine is increased upon fluorination [22]. Facilitated access of protons to the chromophore might be the result of structural rearrangements which can also lead to a reduced shielding from molecular oxygen.…”

Prolegomena zum experimentellen Engineering des genetischen Codes durch Erweiterung seines Aminosäurerepertoires

“…Ein Jahrzehnt später untersuchten Ring und Huber mithilfe der Substitution aller TyrSeitenketten durch (3-F)Tyr die Rolle von Tyr im aktiven Zentrum der b-Galactosidase bei der Katalyse. [112,113] Brooks et al [114] erweiterten 1998 das Repertoire translationsaktiver Tyr-Analoga, indem sie o-Fluortyrosin (47) und 2,3-Difluortyrosin in die Ketosteroid-Isomerase aus Pseudomonas testosteroni einbauten. Der früh von Calender und Berg [115] beschriebene Einbau von l-Dopa (68) in Proteine wurde durch neuere Experimente nicht bestätigt (Abbildung 5).…”