Recombinant expression and isolation of human l-arginine:glycine amidinotransferase and identification of its active-site cysteine residue

Abstract

Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

Weeds, Worms, and More. Papain's Long-Lost Cousin, Phytochelatin Synthase

“…The second is that at least some of the energy required for subsequent condensation of the two substrate molecules is derived from acylation of the enzyme coincident with cleavage of the first substrate. A mechanism for PC synthase analogous to those of Ser proteases (Kraut, 1977), Cys proteases (Kamphuis et al, 1985), and Cys hydrolases (Cheah et al, 1993;Humm et al, 1997aHumm et al, , 1997b) is therefore invoked except that, instead of mediating a dissipative hydrolysis reaction, at least some of the energy required for condensation of the g-Glu-Cys unit from one substrate with the other substrate during the second, PC synthetic, phase of the catalytic cycle is derived from an enzyme oxyester of intermediate energy or an enzyme thioester of high energy formed during the first phase of the cycle.…”

Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study

“…Alternative splicing of the pre-mRNA of AT may explain the occurrence of different forms of AT, namely a cytosolic form of 391 amino acids and a mitochondrial form of 386 amino acids which is generated by cleveage of the 37-residue signal peptide from the 423-amino-acid preprotein (Humm et al, 1997a). The two forms do not arise from a common precursor since the five N-terminal residues of the cytosolic form does not occur in the cleaved signal peptide.…”

Phytochelatin synthase, papain's cousin, in stereo

“…1) (4). What automatically follows is a mechanism for PC synthases analogous to those of serine proteases (11), cysteine proteases (12), and cysteine hydrolases (13,14,15) in which initial nucleophilic attack on the scissile bond of the dipeptidyl donor is by an enzyme hydroxyl-derived oxyanion or thiol-derived thiolate anion. The results of site-directed mutagenic analyses support this proposal and point to a cysteine rather than a serine protease-type mechanism.…”