Functional residues at the active site of bovine brain adenosine deaminase

Abstract

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first‐order kinetics with a second‐order rate constant of 8.9 10‐3 (±1.8 10‐3) M‐1 min‐1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern‐Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground‐ and transitionstate analogs to the enzyme.

Alkylation of Adenosine Deaminase and Thioredoxin by Acrylamide in Human Cell Cultures

“…We did not observe any changes in the ADA activity after acrylamide treatment in vitro. The discrepancy between our data and those of Lupidi et al (1997) might be due to the nature of the reagent: In contrast to propionamide, a small neutral irreversible modifi cation, PCMBS is a relatively big, acidic compound that contains organic mercury. Hg 2+ is an inhibitor of ADA (Franco et al, 1998), and traces of Hg 2+ in PCMBS might also contribute to the observed reduction in the ADA activity.…”

“…On the other hand, there are several evidences that modifi cations of cysteines affect the ADA activity. Lupidi et al (1997) showed that the cysteinemodifying agent PCMBS reduces the ADA activity, and Arrendondo-Vega et al (1998) isolated the ADA mutation G74C from cDNA of patients with severe and delayed onset combined immunodefi ciency. The authors reported further that the mutation G74C shows reduced ADA activity in vitro.…”

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase

“…Therefore proper monitoring of substrate degradation with concomitant product formation are difficult to measure quantitatively. Generally, the adenosine deaminase reaction is assayed spectrophotometrically by measuring the decrease in absorbance at 265 nm (decrease in adenosine concentration) or increase in absorbance at 235 nm (increased formation of inosine) [13,14]. Moreover, in cell lysates or in body fluids the increase in inosine formation cannot be properly studied due to non-specific contributions.…”

A new strategy based on pharmacophore-based virtual screening in adenosine deaminase inhibitors detection and in-vitro study

“…Adenosine deaminase (ADA) is a key enzyme in the purine metabolism that hydrolyse adenosine to inosine irreversibly [1]. This path involves in RNA, DNA, ATP synthesizes, and energy transitions reactions.…”