Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc

Abstract

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV

“…No activity of the C135A, D187A, and C189A mutants was detectable either by the Nessler reaction or by ITC-MIM. The normalized zinc profile of the H139A mutant activity and the kinetic parameters of this mutant, with and without 500 nM Zn 2+ , are collected in Figure 2A and Table 4 , together with normalized zinc profiles and kinetic parameters of the K138A mutant and WT protein, determined with and without their optimal Zn 2+ concentration ( Sliwiak et al, 2024 ). In order to additionally verify the state of occupancy of the zinc binding site, titrations of the muteins (not pretreated with any chelator) with Zn 2+ were performed microcalorimetrically, leading to K D determination for the K138A and D187A mutants only ( Figure 2B ; Table 5 ).…”

“…The optimal zinc concentration for WT ReAV activity appeared to be 2.5 μM ( Figure 2A ), which is close to the K D of zinc binding (∼3 μM) by this protein. This optimal zinc concentration boosted the ReAV activity by more than 50% ( Sliwiak et al, 2024 ). We could also observe that crippling the zinc coordination by the K138A mutation shifted this Zn 2+ optimum to 10 μM, and eliminated the slight inhibitory effect of 100 μM zinc concentration observed for WT ReAV (100 μM Zn 2+ is still strongly stimulative for K138A).…”

“…The relative activity expressed as the percentage of enzyme activity without zinc supplementation was calculated and plotted to compare the shape of the zinc profiles and the level of response to Zn 2+ . The kinetic parameters without and with optimal zinc concentration in 10 mM Tris-HCl pH 9.0 buffer were determined by the multiple injection isothermal titration calorimetry method (ITC-MIM), as described in Sliwiak et al (2024) . Briefly, the average enthalpy of total conversion of L-Asn was determined by four injections of 2 µL of 10 mM L-Asn (Sigma-Aldrich) into the reaction cell containing 2–10 µM of the enzyme, using 150–600 s intervals.…”

“…It was established that Zn 2+ binding is highly selective ( Loch et al, 2021 ), with K D of ∼3 μM. Moreover, it was shown that low and optimal zinc concentration can boost the activity of ReAV by over 50%, while simultaneously improving the substrate specificity, indicating its role in substrate recognition ( Sliwiak et al, 2024 ). There is also an indirect connection between Cys135 from the metal coordination sphere and an oxidized Cys249, which is involved in a network of H-bonds comprising the entire active site area ( Figure 1B ).…”

“…While wild-type (WT) ReAV is a fast enzyme (K M 2.1 mM, k cat 603 s −1 , k cat /K M 294 mM −1 ·s −1 at pH 9.0) ( Sliwiak et al, 2024 ), its substrate affinity is by ca. two orders of magnitude too low for application as an antileukemic drug.…”