Introduction of a Phe / Tyr-Mutation and Simultaneous Removal

Aldolase

The steady-state kinetic measurements for theTacTAL variants with enhanced aldolase activity were performed as described in section 1.6.4.2. The used coupled spectrophotometric assay detects the formation of the first product glyceraldehyde 3-phosphate (GA3P). The both activities (transaldolase and aldolase) result in the formation of this product. Therefore, in presence of acceptor substrate E4P it cannot be discriminated between transaldolase and aldolase reaction.

However, in absence of acceptor substrate only the aldolase activity can occur. The measured activity was plotted against the substrate concentration (figure 2.2.17) and fitted according to the Michaelis-Menten-equation (eq. 1.6.2).

TacTALwtcatalyzes the aldolytic cleavage of the donor substrate F6P as a very slow side reaction.

The turnover number for this reaction was determined tok_cat=∼(2.25±0.01)·10⁻³s⁻¹(data not shown). The introduction of a tyrosine residue in the active site ofTacTAL giving theTacTALF132Y

variant results in an increase of the aldolase activity in terms ofk_catby two orders of magnitude compared to wild type (table 2.2.2). The second mutation of Glu⁶⁰to glutamine enhanced this activity by an additional factor of three. Further introduction of bulky hydrophobic residues in

Figure 2.2.17: Dependency of the aldolase ac-tivity (cleavage of F6P into GA3P and DHA) of TacTAL variants on F6P concentration.

The steady-state measurements were performed at 30^◦C. The substrate concentration was varied (0.1 – 100 mM). The rectangles, triangles and circles indicate the measured aldolase activity ofTacTAL_FSA-mimic (125^μg/^mL), TacTAL_F132Y (120^μg/^mL) and TacTALE60Q/F132Y (35^μg/^mL), respectively. The Michaelis-Menten fits (eq. 1.6.2, solid line) are depicted by green, blue and red lines, respectively.

Table 2.2.2: Overview of macroscopic kinetic constants for aldolase activity (cleavage of F6P into GA3P and DHA) ofEcFSAwt, TacTALwt andTacTAL variants. The margin of error is given as the standard deviation of the fitted data (sets of triplicates). nd=not detectable. *taken from Schneideret al. (2008), **published in Sautneret al. (2015), ***data provided by Lietzow (2015).

Protein kcat(s⁻¹) K^app_M (mM) ^kcat/^KappM (s⁻¹M⁻¹)

TacTALwt (2.25 ± 0.01)·10⁻³ nd nd

TacTALFSA-mimic*** (2.41 ± 0.02)·10⁻² 2.41 ± 0.09 10.00

TacTAL_F132Y** 0.24 ± 0.01 12.30 ± 1.50 19.51

TacTALE60Q/F132Y** 0.62 ± 0.01 1.50 ± 0.10 413.33

EcFSAwt* 1.30 ± 0.30 12.00 ± 3.00 108.33

the „down“ site of the active site (Ser⁵⁸→Phe, Asn¹⁰⁸→Leu and Ser¹³⁰→Ala) results in a 5x-variant (TacTAL_FSA-mimic) with a greatly impaired aldolase activity compared to the double variant (TacTALE60Q/F132Y). The aldolase activity of theTacTALFSA-mimicvariant is one order of magnitude lower than that of theTacTAL_F132Y variant. This result is surprising since theTacTAL_FSA-mimic variant reflects the situation in the active site ofEcFSA better than the double variant regarding the relative positions of particular amino acid residues in the active site. However, theTacTAL_FSA-mimic variant still shows one order of magnitude higher aldolase activity compared toTacTALwt. In order to estimate the transaldolase activity (reversible transfer of a DHA-unit from the donor F6P to the acceptor E4P) of the variants, the steady-state measurements were performed in presence of 2 mM E4P. The presence of the acceptor substrate (E4P) does not influence the activity of the TacTAL_F132Y variant and inhibits only slightly the activity of the TacTAL_E60Q/F132Y variant (decrease in activity by∼10 %). In other words, the transaldolase activity of the variants is either lower than the respective aldolase activity or equal to that. Thus, the transaldolase activity of the

Table 2.2.3: Overview of macroscopic kinetic constants for transaldolase activity (reversible transfer of a DHA-unit from F6P to E4P) ofTacTAL_wtand variants. The margin of error is given as the standard deviation of the fitted data (sets of triplicates). ND=not determined. *taken from Lehwess-Litzmann(2011), **published in Sautneret al. (2015), ***data provided by Lietzow (2015).

Protein kcat(s⁻¹) K^app_M (mM) ^kcat/^KappM (s⁻¹M⁻¹)

TacTALwt* 13.86 ± 0.65 2.30 ± 0.36 6026.1

TacTAL_FSA-mimic*** ≤ 2.82·10⁻² ND ND

TacTALF132Y** ≤ 0.24 ND ND

TacTALE60Q/F132Y** <0.62 ND ND

TacTAL_E60Q* 0.12 ± 0.01 0.49 ± 0.13 244.90

variants is much lower than the corresponding activity of the wild type protein (table 2.2.3). The addition of E4P slightly stimulates the activity of theTacTALFSA-mimic variant (increase in activity by∼10 %). This activation effect in presence of E4P could mean that theTacTAL_FSA-mimic variant catalyzes the transaldolase reaction slightly better than the aldolase reaction and could be a hint for the impaired protonation of the carbanion/enamine intermediate in this variant compared to the TacTALE60Q/F132Yvariant, which is essential for the aldolase reaction.

The apparent Michaelisconstant (K^app_M ) for the aldolase reaction ofTacTALF132Yis approximately equal to that of EcFSA_wt (table 2.2.2). This value is fivefold higher than the corresponding constant for the transaldolase reaction ofTacTALwt. The introduction of the second mutation (Glu⁶⁰→Gln) leads to lowerK^app_M (∼8x lower compared to the single-mutation variant) as well as to a higher turnover number. Both effects give aTacTAL variant with a higher catalytic efficiency (^kcat/^KappM =413.33 s⁻¹M⁻¹) than in case of the native fructose 6-phosphate aldolase fromE. coli (^kcat/^KappM =108.33 s⁻¹M⁻¹, (Schneideret al., 2008)). TheK^app_M value for F6P conversion catalyzed by theTacTAL_FSA-mimicvariant is similar to that ofTacTAL_wtbut because of the low turnover number the catalytic efficiency of this variant is one order of magnitude lower compared toEcFSAwt. In other words, the introduction of a single Phe→Tyr mutation in the active site of transaldolase (E. coli: Phe¹⁷⁸, human: Phe¹⁸⁹,Thermoplasma acidophilum: Phe¹³²) is the minimum requirement to induce the aldolase activity (Lehwess-Litzmann, 2011; Sautneret al., 2015; Schneideret al., 2008). The additional mutation of the original general acid-base catalyst ofTacTAL (Glu⁶⁰) to glutamine results in a variant with a greatly improved catalytic efficiency towards the aldolase reaction. This double-variant (TacTAL_E60Q/F132Y) shows the best results in terms ofk_cat,K^app_M and

k_cat/^KappM for the aldolase activity compared to other variants introduced in this section. Further introduction of a hydrophobic surface in the „down“ site of theTacTAL’s active site (mutations:

Ser⁵⁸→Phe, Asn¹⁰⁸→Leu and Ser¹³⁰→Ala) to mimic the situation in the active site ofEcFSA_wt results in the worst „aldolase“ analyzed in this work.

2.2.3.2 The Active Site ofTacTALFSA-mimicVariant Shows a Different pKaBehavior