L-天冬酰胺酶,分为三个结构类,催化L-天冬酰胺水解为L-天冬氨酸和氨。第三类,ReAIV和ReAV的成员,编码在固氮根瘤菌等的基因组中,有相同的褶皱,活动站点,和第四纪结构,尽管序列同一性低。在目前的工作中,我们检查了这种差异的生化后果。在37°C的天冬酰胺水解中,ReAIV的效率几乎是ReAV的两倍,有了动力学KM,Kcat参数(在最佳缓冲剂中测量)为1.5mM,770s-1和2.1mM,分别为603s-1。ReAIV的活性在45°C-55°C时具有最佳温度,而ReAV的活性,在37°C时达到最佳状态后,在45°C时急剧下降。两种同工型的活性提高了32%或56%,通过低和最佳的锌浓度,ReAIV比ReAV强三倍,正如1.2和3.3μM的KD值所反映的那样,分别。我们还证明了Lys→Ala点诱变对锌结合的扰动会大大降低酶活性,但也会改变对锌的反应方式。我们还检查了不同二价阳离子对活性的影响,动力学,和两种同工型的稳定性。似乎Ni2+,Cu2+,Hg2+,和Cd2具有按以下顺序(从最强到最弱的抑制剂)抑制两种同工型的潜力:Hg2>Cu2>Cd2>Ni2。ReAIV对Cu2+和Cd2+更敏感,而ReAV对Hg2+和Ni2+更敏感,如IC50值所示,熔化扫描,以及对底物特异性的影响。低浓度的Cd2+提高了两种同工型的底物特异性,表明其在底物识别中的作用。在ReAIV的情况下,对Hg2+进行了相同的观察。ReAV同工型的活性对Cl-阴离子较不敏感,如NaCl的IC50值所反映的,ReAV相对于ReAIV高8倍。两种同工型的未发现的互补特性有助于我们更好地理解ReAV酶的诱导性。
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.