Mutations at Positions 153 and 328 inEscherichia coliAlkaline Phosphatase Provide Insight Towards the Structure and Function of Mammalian and Yeast Alkaline Phosphatases

Murphy, Jennifer E.; Tibbitts, Thomas T.; Kantrowitz, Evan R.

doi:10.1006/jmbi.1995.0576

Cited by 94 publications

(100 citation statements)

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“…These water molecules are further coordinated and stabilized by other amino acid residues including Asp-153. The D153H mutation in ECAP was shown to destabilize the octahedral Mg coordination in favor of a tetrahedral one and resulted in an enzyme that had reduced Mg 2ϩ affinity and increased Zn 2ϩ affinity and was significantly activated by Mg 2ϩ (21). This is strongly reminiscent of the behavior of the IAP isozyme (1) but not of human PLAP, which binds Mg 2ϩ tightly and in which the further addition of Mg 2ϩ does not increase activity.…”

Section: Mutagenesis Of Metal Ligands In Plap-mentioning

confidence: 70%

“…The substitution of D153H and K328H in ECAP produced enzymes with kinetic properties similar to those of mammalian APs. For example, the D153H/K328H double ECAP mutant displayed a 5.6-fold higher k cat and a 30-fold higher K m , a decrease in heat stability, and a shift in pH optimum to alkaline pH values (21). We constructed the reciprocal mutations, i.e.…”

Section: Mutagenesis Of Metal Ligands In Plap-mentioning

confidence: 99%

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Function Assignment to Conserved Residues in Mammalian Alkaline Phosphatases

Kozlenkov¹,

Manes²,

Hoylaerts³

et al. 2002

Journal of Biological Chemistry

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We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k cat and increased K m when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxylterminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k cat , a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors L-Phe and L-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.Alkaline phosphatases (APs 1 ; EC 3.1.3.1) are a family of dimeric metalloenzymes catalyzing the hydrolysis of monoesters of phosphoric acid (1). APs also catalyze a transphosphorylation reaction in the presence of large concentrations of phosphate acceptors. APs occur widely in nature and are found in many organisms from Escherichia coli to man. APs are homodimeric enzymes, and each catalytic site contains three metal ions (two Zn ions and one Mg ion) that are necessary for enzymatic activity (2). Whereas the main features of the catalytic mechanism are conserved between mammalian APs and the E. coli enzyme, there are important differences. Mammalian APs (a) have higher specific activity and K m values, (b) have a more alkaline pH optimum, (c) are inhibited by L-amino acids and peptides through an uncomp...

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Section: Mutagenesis Of Metal Ligands In Plap-mentioning

confidence: 70%

Section: Mutagenesis Of Metal Ligands In Plap-mentioning

confidence: 99%

Function Assignment to Conserved Residues in Mammalian Alkaline Phosphatases

Kozlenkov¹,

Manes²,

Hoylaerts³

et al. 2002

Journal of Biological Chemistry

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“…Using human placental AP as a paradigm for mammalian APs, we and others have established that residues within a surface loop unique to mammalian APs are responsible for the differential uncompetitive inhibition by L-amino acids (9 -12), the heat stability properties (13), and protein-protein interaction specificities exhibited by some mammalian APs (13). A major property of APs that remains to be explained in terms of structure is the large variability in catalytic activity displayed by mammalian APs, which have 10 -100-fold higher k cat values than E. coli AP (14). Because among mammalian APs, the intestinal isozyme has the highest specific activity, the bovine intestinal APs (IAPs) represent a potentially useful system for addressing this question.…”

Section: ؊1mentioning

confidence: 99%

Genetic Complexity, Structure, and Characterization of Highly Active Bovine Intestinal Alkaline Phosphatases

Manes¹,

Hoylaerts²,

Müller³

et al. 1998

Journal of Biological Chemistry

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Mammalian alkaline phosphatases (APs) display 10 -100-fold higher k cat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, ϳ3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, ϳ8,000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH 2 -terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wildtype and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed average k cat values of 1800, 5900, 4200, and 6100 s ؊1 , respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in k cat . Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its k cat to 1300 s ؊1 , while the converse mutation, i.e. D322G, in bIAP I increased its k cat to 5800 s ؊1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex.

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“…Mutagenesis of the ECAP residue corresponding to TAP H135 (ECAP D153H) results in a tetrahedral M3 site, which is occupied by a Zn 2þ . 15 The enzyme has decreased metal affinity, but at high concentrations of Mg 2þ and combined with a mutation at D330, the enzyme is 17-fold more active than wild-type. 11,16 TAP is a typical cold-adapted enzyme: it has naturally high catalytic activity but it is relatively unstable at high temperatures.…”

Section: Introductionmentioning

confidence: 99%

Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases

Koutsioulis¹,

Lyskowski²,

Mäki³

et al. 2009

Protein Science

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Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology.

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Mutations at Positions 153 and 328 inEscherichia coliAlkaline Phosphatase Provide Insight Towards the Structure and Function of Mammalian and Yeast Alkaline Phosphatases

Cited by 94 publications

References 0 publications

Function Assignment to Conserved Residues in Mammalian Alkaline Phosphatases

Function Assignment to Conserved Residues in Mammalian Alkaline Phosphatases

Genetic Complexity, Structure, and Characterization of Highly Active Bovine Intestinal Alkaline Phosphatases

Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases

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