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Common-Type Acylphosphatase

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Common-Type Acylphosphatase Biochem. J. (1997) 327, 177–184 (Printed in Great Britain) 177 Common-type acylphosphatase: steady-state kinetics and leaving-group dependence Paolo PAOLI*, Paolo CIRRI*, Lucia CAMICI*, Giampaolo MANAO*, Gianni CAPPUGI*, Gloriano MONETI† Giuseppe PIERACCINI†, Guido CAMICI* and Giampietro RAMPONI*1 *Dipartimento di Scienze Biochimiche, Universita' di Firenze, Viale Morgagni 50, Firenze, Italy, and †Centro Interdipartimentale di Servizi di Spettrometria di Massa, Universita' di Firenze, Italy ") A number of acyl phosphates differing in the structure of the acyl does it catalyse H# O–inorganic phosphate oxygen exchange. It moiety (as well as in the leaving-group pKa of the acids produced seems that no phosphoenzyme intermediate is formed in the in hydrolysis) have been synthesized. The K and V values for catalytic pathway. Furthermore, during the enzymic hydrolysis m max ") the bovine common-type acylphosphatase isoenzyme have been of benzoyl phosphate in the presence of O-labelled water, only ") measured at 25 mC and pH 5.3. The values of kcat differ widely in inorganic phosphate (and not benzoate) incorporates O, relation to the different structures of the tested acyl phosphates: suggesting that no acyl enzyme is formed transiently. All these linear relationships between log kcat and the leaving group pKa, findings, as well as the strong dependence of kcat upon the leaving as well as between log kcat}Km and the leaving-group pKa, were group pKa, suggest that neither a nucleophilic enzyme group nor observed. On the other hand, the Km values of the different general acid catalysis are involved in the catalytic pathway. The substrates are very close to each other, suggesting that the enzyme is competitively inhibited by Pi, but it is not inhibited by phosphate moiety of the substrate is the main chemical group the carboxylate ions produced during substrate hydrolysis, interacting with the enzyme active site in the formation of the suggesting that the last step of the catalytic process is the release enzyme–substrate Michaelis complex. The enzyme does not of Pi. The activation energy values for the catalysed and catalyse transphosphorylation between substrate and con- spontaneous hydrolysis of benzoyl phosphate have been de- centrated nucleophilic acceptors (glycerol and methanol); nor termined. INTRODUCTION dimensional structure of muscle isoenzyme has been determined by NMR techniques ([12], and citations herein). Recently, Acylphosphatase is a low-molecular-mass enzyme that catalyses crystals of the CT-isoenzyme have been produced [13] and the the hydrolysis of the carboxy-phosphate bond, and it is wide- three dimensional structure of this isoenzyme has been deter- spread in all vertebrate tissues [1]. There is considerable evidence mined by X-ray crystallography [14]. The overall structure of to suggest that the enzyme is involved in the control of ion-pump CT-acylphosphatase (a basic protein that consists of 98 amino- activities, since it is able to hydrolyse the aspartyl-phosphate acid residues) reveals a very compact protein, consisting of five- bonds that are produced during the action of membrane Na+-, # stranded mixed-sheet with two helices running parallel to the K+- ([2], and citations herein), and Ca +-pumps ([3], and citations sheet. The sheet is slightly curved with a right-handed twist, and herein). The enzyme is also implicated in the control of glycolytic the helices interact with one side of the sheet forming a compact flow, since it hydrolyses 1,3-bisphosphoglycerate, releasing Pi core [14]. Site-directed mutagenesis experiments (performed with and maintaining ADP concentrations at levels suitable to sustain MT-acylphosphatase) have suggested that Arg-23 and Asn-41 high glycolytic flow [4,5]. Previous papers demonstrated that the are essential residues [15,16], Arg-23 being involved in the binding thyroid hormones enhance acylphosphatase expression [6,7]. of the substrate phosphate moiety [14]. This paper deals These findings suggest that part of the excess of heat production with steady-state kinetic studies performed on the CT- in hyperthyroidism is caused by the increased levels of acylphosphatase isoenzyme. acylphosphatase, which has a role in a futile cycle involving 1,3- bisphosphoglycerate [8] and in the uncoupling of Na+-, K+- and # Ca +-pumps [2,3]. Two acylphosphatase isoenzymes are expressed in animals in MATERIALS AND METHODS a tissue-specific manner [9,10], which are named the muscle type Materials (MT) and the organ common (or erythrocyte) type (CT), since the former is highly expressed in skeletal muscle and heart, The CT-isoenzyme was purified from bovine testis as previously ") whereas the latter is expressed in all tissues [10], although its described [11]. [ O]water at 97% isotope enrichment was pur- $# expression is particularly high in erythrocytes, brain and testis. chased from Cambridge Isotope Laboratories. [ P]Pi (8500 Both isoenzymes have been isolated from a number of vertebrate Ci}mmol) was purchased from NEN. All other reagents were tissues and sequenced ([11], and citations herein). The three- the purest commercially available. Abbreviations used: BCA, bicinchoninic acid; MT, muscle type; TMS, trimethylsilyl; CT, organ common type; PTPase, phosphotyrosine protein phosphatase. 1 To whom correspondence should be addressed. 178 P. Paoli and others Acyl phosphates absorbance was read at 510 nm 15 min later. Controls without enzyme were prepared and incubated as described. The V and Acyl phosphates having differing acyl groups were synthesized. max K (means S.E.) were calculated by fitting the initial rate data Benzoyl phosphate and 2-methoxybenzoyl phosphate were pre- m ³ to the Michaelis–Menten equation with the non-linear regression pared as previously described [17,18]. Acetyl phosphate, pro- program Fig. P (Biosoft). All initial rate measurements were pionyl phosphate, and butyryl phosphate were synthesized as carried out at least in triplicate. follows: 40 mmol of each acyl anhydride were slowly added to a mixture of 40 mmol of phosphoric acid dissolved in 48 ml of 30% (v}v) pyridine, previously chilled in ice. The mixture was Protein assay stirred for about 1 h, then 120 mmol of LiCl dissolved in 12 ml of water was added. The acyl phosphates were precipitated by Protein concentration was determined by the bicinchoninic acid adding cold ethanol or, in some cases, ethanol–acetone mixtures. (BCA) kit method (Sigma), using BSA as standard. Phenylacetyl and p-nitrobenzoyl phosphates were prepared using a similar but slightly modified method, since the corresponding Benzoyl phosphate enzymic hydrolysis in [18O]water anhydrides were not commercially available. Anhydrides were then synthesized from free acid and acyl chlorides as follows: Benzoyl phosphate (1 mM final concentration) was dissolved in ") 40 mmol of each free acid and 40 mmol of triethylamine were [ O]water, and pH was adjusted to 5.5. A small amount of dissolved in 8 ml of anhydrous tetrahydrofuran. The solutions acylphosphatase was then added, and the mixture was incubated were chilled in ice, and 40 mmol of the corresponding acyl for 40 min at room temperature to achieve complete hydrolysis. chloride was slowly added under stirring. The precipitates formed A portion of 50 µl was withdrawn, transferred in a screw-cap during the reactions were discarded by filtration. The acid conical vial and dried. Successively, sample derivatives were anhydrides in the mixture were concentrated under vacuum and obtained for GLC–MS analysis, as described below. added to the phosphate–pyridine mixture as described for acetyl phosphate, propionyl phosphate and butyryl phosphate syn- 18 thesis. Product yields ranging from 30% to 50% were obtained. Inorganic phosphate–medium water O exchange Some acyl phosphates, such as acetyl phosphate, propionyl Mes (10 mM final concentration) and Pi (100 mM final con- phosphate and butyryl phosphate, were purified by repeated centration) were dissolved in water and the pH was adjusted to fractional precipitation with ethanol, whereas phenylacetyl phos- 5.5 with NaOH. Then 100 µl samples were withdrawn, transferred phate and p-nitrobenzoyl phosphate were purified by preparative to a small screw-cap conical vial and dried. The residue was ") reversed-phase chromatography using a C18 preparative bulk- dissolved in 100 µlof[ O]water, and 1 µl of acylphosphatase (15 packing phase (55–105 µm, Waters, U.S.A.) following the units; the unit is defined as the amount of enzyme that catalyses procedure previously described for the purification of 2-meth- the hydrolysis of 1 µmol of benzoyl phosphate at 25 mC and oxybenzoyl phosphate [18]. The final products were analysed for pH 5.3) was added. The mixture was incubated at room tem- acyl phosphate bond content using the hydroxylamine–ferric perature. Aliquots of 1 µl were withdrawn at various incubation chloride photometric method and calibration curves described in times and diluted with 100 µl of acetonitrile contained in a small the following section [19]. The free Pi content in the acyl screw-cap conical vial. The mixture was dried, and derivatives of phosphates was assayed by the method of Baginski et al. [20]. $# inorganic phosphate were formed for GLC–MS analysis, as Benzoyl [ P]phosphate was synthesized as previously described $# described below. In order to check the performance of the [21] using [ P]Pi and benzoic anhydride. method, a parallel experiment with calf intestine alkaline phos- phatase was performed [in this experiment, the pH of the Enzyme assay incubation mixture (100 µl) was adjusted to 9.0, and 5 enzyme The 2-methoxybenzoyl and benzoyl phosphatase activities were units were added (the unit is defined as the amount of enzyme determined by continuous spectrophotometric assays as pre- that catalyses the hydrolysis of 1 µmol of p-nitrophenyl phos- viously described [18,22]. p-Nitrobenzoyl phosphatase activity phate at 25 mC and pH 9.8)]. It is well known that alkaline was assayed by a similar method following the time-dependent phosphatase forms a covalent enzyme–phosphate intermediate during its catalytic process before releasing Pi [23], and thus is absorbance change owing to the absorption difference between ") the substrate and its hydrolysis product at 313 nm.
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