Structural aspects of human prostatic acid phosphatase and fast atom bombardment mass spectrometric analysis of Escherichia coli L-threonine deaminase

Ross Edward Davidson, Purdue University

Abstract

Human prostatic acid phosphatase (E.C. 3.1.3.2) is a dimeric enzyme composed of two identical non-covalently linked subunits of MW $\approx$ 47,000 as determined by SDS gel electrophoresis. The enzyme has many different isoelectric forms. Some of these isoelectric forms were shown to be due to post-translational processing of the enzyme at the C-terminal end. In addition, some of the forms were shown to be a result of deamidation of glutamine or asparagine side chains. Two previously published amino acid sequences of the enzyme were shown to contain errors. One of these had mis-identified cysteine-340 as a valine. The determination of the correct residues permitted the assignment of the disulfide bonds of HPAP. Three disulfides were identified, between cysteine residues 129 and 340, 183 and 281, as well as 315 and 319. Due to the high degree of homology of human lysosomal acid phosphatase (HLAP) with HPAP, HLAP was predicted to have the same disulfide bonding pattern as HPAP. Iodination of two tyrosine residues, at amino acid residues 52 and 57 confirmed a prediction that these two residues are located on the protein surface. The sequence region including and surrounding these tyrosines was shown to contain an epitope that binds to rabbit polyclonal antibodies raised against native HPAP. The modification of an arginine residue by a relatively small (5X) excess of radiolabeled phenylglyoxal permitted the identification of arginine-54 as an active site residue. The reaction was highly selective and relatively rapid. Arginine-54 corresponds to the first residue in an Arg-Lys-Arg-Tyr-Arg sequence shown to have conservative homologs in human and rat lysosomal acid phosphatases, a protein tyrosine phosphatase, and creatine kinase M chain. In the case of phosphatases, this region seemed to exist only in phosphatases that are tartrate-inhibited. No example of a tartrate-resistant enzyme with this sequence region was found. Trapping of an enzyme phosphohistidine intermediate using $\sp{32}$P-labeled p-nitrophenyl phosphate, followed by gas phase sequencing of a labeled peptide, identified histidine-12 as an active site residue. This histidine is contained in a RHGXRXP region with homologies in many different phosphatases as well as with creatine kinase. The sequence of a recombinant L-threonine deaminase was verified by FAB-MS. A single disulfide bond between monomers was located between the cysteine-378 residues of each monomer by FAB-MS. The location of the pyridoxal 5$\sp\prime$-phosphate binding site at lysine-62 was also determined by FAB-MS. This binding region retains a high degree of homology in many of the threonine deaminases.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry

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