Fujio Nagashima

Pyridoxal phosphate-binding site in enzymes; reduction and comparison of sequences.

Publisher Summary This chapter illustrates pyridoxal phosphate-binding site in enzymes. In pyridoxal phosphate-dependent enzymes, the formyl group at position 4 of pyridoxal-P forms an aldimine bond with the ɛ -amino group of a specific lysyl residue. Involvement of a single particular lysyl residue indicates that there is a specific domain structure favorable for stabilizing the aldimine bond which, otherwise, is readily hydrolyzed. X-Ray analyses of both cytosolic and mitochondrial isoenzymes of aspartate aminotransferase, representative pyridoxal-P-dependent enzymes, provide details of interaction between pyridoxal-P and the enzyme side chains in the coenzyme-accommodating active site. Only the Re side of the aldimine bond is open to solvent, the Si face being shielded from solvent. This stereospecific status of the bound coenzyme has been remarkably identical among a variety of pyridoxal-P-dependent enzymes. At neutral pH, pyridoxal-P absorbs at 390 nm, which shifts more or less to a longer wavelength (410-430 nm) upon the formation of an aldimine linkage with amino compounds or apoenzymes. Pyridoxal-P-aldimine bond is reduced by borohydride or borocyanohydride under mild conditions. The reduction renders the aldimine bond highly stable to hydrolysis. The resulting reduced enzyme is fragmented by either proteolytic or chemical cleavage, followed by isolation of a phosphopyridoxyl peptide and its structural determination.

Affinity chromatography of hepatic glutathione S-transferases on ω-aminoalkyl sepharose derivatives of glutathione

Rat liver glutathione S-transferases (RX: glutathione R-transferase, EC 2.5.1.18) were found to adsorb S-carbamidomethyl glutathione linked to Sepharose CL-4B via lysyl or aliphatic diamine spacers of various carbon chain lengths (-NH-(CH2)n-NH-, n = 2, 4, 5, 6, 8 and 10). Proteins were eluted specifically by reduced glutathione. The affinity of the enzymes for the adsorbent increased with increase in the carbon chain length of aliphatic diamine spacers used. Adsorbent having a free carboxyl group within the spacer moiety had high capacity and was specific for glutathione S-transferases. The transferases were specifically eluted from the column in high yield by low concentrations of glutathione. Enzymes purified by the lysyl spacer adsorbent were homogeneous in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and contained most of the hepatic glutathione S-transferase isozymes in isoelectric focusing. Oxidized glutathione and S-methyl glutathione were equally effective as reduced glutathione in eluting glutathione S-transferases from the adsorbent, but gamma-glutamylcysteinylglycineamide or gamma-glutamylcysteinylglycine-1-methyl ester were not effective. These data suggested that the free carboxyl group of glycyl moiety of glutathione might also be important for the specific binding of the transferases to this adsorbent.

Structure-Function Relation of the Presequence of a Precursor to Pig Mitochondrial Aspartate Aminotransferase

The efficiencies of mitochondrial import and processing of some variants of pre-mitochondrial aspartate aminotransferase (pre-mAspAT) with mutations within its presequence were tested. The amino-terminal portion is essential for mitochondrial uptake and processing. The presence of two positively charged residues, Hisl8 and Arg28, may not be requisite for the uptake, but the processing efficiency was reduced by the single amino acid change, Arg28 to a leucyl residue.

Kinetic Studies on Binding of Mitochondrial and Cytosolic Aspartate Aminotransferases with Suicide Substrate, Gostatin

Binding of pig heart mitochondrial and cytosolic aspartate aminotransferases (m-AST and c-AST) with a specific inhibitor, gostatin (5-amino-2-carboxy-4-oxo-1,4,5,6-tetrahydropyridine-3-acetic acid) was studied kinetically, by monitoring the spectral change with a micro-stopped-flow apparatus and by following the inactivation of the enzyme activity. No appreciable difference in kinetics was observed between m-AST and c-AST. Any positive evidence for the existence of spectrophotometrically distinguishable intermediate was not obtained. Under the condition studied, the second-order rate constant of spectral change was about 1.5 to 2 times as large as that of inactivation.

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide.

Reaction of N‐bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N‐bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.