Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme

Abstract

The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

“…Despite the great number of proteins that bind compounds containing a phosphoryl group~Copley & Barton, 1994!, stabilization by phosphate has been shown for relatively few proteins and enzymes thus far, such as, for example, acid phosphatase~Cashikar & Rao, 1996!, RNAse~Meiering et al, 1991!, the apoenzyme of aspartate aminotransferase~Iriarte et al, 1985;Martinez-Liarte et al, 1992!, andmaltodextrin phosphorylase from E. coli~Grießler et al, 2000!. With the exception of maltodextrin phosphorylase for which phosphate prevents the electrostatically driven aggregation of a reversibly inactivated enzyme dimer, stabilization of the three other enzymes was due to binding of phosphate in the active site, and therefore most significant in the case of the enzyme activity. The mechanism by which phosphate increases the stability of StP against denaturation is clearly different from these examples, and the magnitude of the stabilizing effect appears to be without precedent in the literature.…”

Sulfate ion interaction with ‘anion recognition’ short peptide motif at the N-terminus of an isolated helix: A conformational landscape

“…This includes substrate and cofactor binding (Barrett et al, 1998;Cobessi et al, 1999), phosphate transportation (Ledvina et al, 1996), electron transfer and antioxidant activity (Abdullaev et al, 2002), recognition of enzymes and nucleotides binding (Wierenga et al, 1984;Symmons et al, 2000;Ramakrishnan et al, 2002), providing extra stability to proteins (Martinez-Liarte et al, 1992;Meiering et al, 1991), affecting pK a of histidine (Smith et al, 1989) and others (Dealwis et al, 1998). In anion bound form the anion recognition site of the protein segment participate in formation of a ''functional surface" where anions usually bind to the main chain atoms of the protein segment and the interaction is mediated by specific positioning of the protein backbone (Chakrabarti, 1993;Denessiouk et al, 2001Denessiouk et al, , 2005Watson and Milner-White, 2002).…”

Phosphate Recognition in Structural Biology

“…Binding of a phosphoryl group is essential to a myriad of biological processes ranging from metabolism and biosynthesis, to gene regulation, signal transduction, muscle contraction, and antibiotic resistance. Phosphate binding confers extra stability to enzymes such as Asp aminotransferase [7] and induces iron deposition in apoferritin. [8] Protein phosphorylation is central to regulating transmembrane and intracellular signal transduction pathways.…”