Crystal Structure and Allosteric Regulation of the Cytoplasmic Escherichia coli l-Asparaginase I

Abstract

AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell.

Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties

“…In other words, just as observed for the low K m bacterial enzymes, binding of ligand to gpASNase1 does not induce a profound quaternary conformational change (for detailed substrate-induced conformational changes see below). This could be due to the fact that EcI is an allosteric enzyme requiring a second molecule of Asn to bind to a site distant from the active site (9). gpASNase1 follows classic Michaelis-Menten kinetics, and no cooperativity was observed.…”

“…Escherichia coli has several L-asparaginases, but only one of them, the type II L-asparaginase, denoted here as EcII (gene name ansB), has the prerequisite low Asn K m property (K m of 11.5-15 M (7,8)). Interestingly, the type I E. coli L-asparaginase, denoted as EcI (gene name ansA) is structurally similar to EcII but has a K m in the millimolar range (9). EcII was approved by the Food and Drug Administration for the treatment of certain blood cancers such as ALL in 1978 under the brand name Elspar.…”

Avaliação da atividade e resistência à clivagem proteolítica de L-asparaginases recombinantes obtidas por reação em cadeia da polimerase propensa a erro

“…Estas enzimas estão presentes em bactérias, fungos, plantas e mamíferos; nos quais, sua ação primordial é formar aspartato, que pode ser transaminado em oxaloacetato, o qual participa do ciclo do ácido tricarboxílico, ou ser convertido em fumarato, durante o ciclo da uréia (YUN et al, 2007).…”

“…Já a EcA II é expressa no periplasma, apenas em situações anaeróbicas e de escassez de nutrientes. Possui alta afinidade pelo substrato (K M = 10-15 µM), a qual é essencial para que haja a captação da pouca asparagina presente no meio ambiente (YUN et al, 2007).…”

“…Outra diferença entre as duas L-asparaginases de E. coli é que apenas a EcA II possui propriedades antileucêmicas, tanto in vitro quanto in vivo, e é utilizada no tratamento da LLA (BOREK et al, 2014;LABROU;PAPAGEORGIOU;AVRAMIS, 2010;YUN et al, 2007 Na clínica, após administração intravenosa da L-asparaginase, ocorre rápida depleção plasmática do aminoácido L-asparagina e, em menor escala, do aminoácido glutamina (PIETERS et al, 2011). Diversas células saudáveis do organismo humano possuem asparagina sintetase e são capazes de sintetizar a Lasparagina, a partir da L-glutamina (CHAN et al 2014;NARTA;KANWAR;AZMI, 2007).…”