No abstract

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.

A combination of limited tryptic proteolysis, reverse phasehigh performance liquid chromatography, Edman degradative sequencing, amino acid analysis, and fast-atom bombardment mass-spectrometry was used to remove and identify the first 14 to 18 N-terminal amino acid residues of the large subunit of higher plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas relnhardtii, Marchantia polymorpha, pea (Pisum satvum), tomato (Lycoperskon esculentum), potato (Solanum tuberosum), pepper (Capskum annuum), soybean (Glycine max), petunia (Petunia x hybrida), cowpea (Vigna sinensis), and cucumber (Cucumis sativus) plants. The N-terminal tryptic peptide from acetylated Pro-3 to Lys-8 of the large subunit of Rubisco was identical in all species, but the amino acid sequence of the penultimate N-terminal tryptic peptide varied. Eight of the 10 species examined contained a trimethyllysyl residue at position 14 in the large subunit of Rubisco, whereas Chlamydomonas and Marchantla contained an unmodified lysyl residue at this position.Rubisco (EC 4.1.1.39) is a large hexadecameric protein, with 8 large and 8 small subunits, that catalyzes the fixation of atmospheric CO2 during photosynthesis (2). The large subunit is encoded by chloroplast DNA (8) and the small subunit by nuclear DNA (13). The small subunit is synthesized as a precursor with an N-terminal transit sequence that targets the polypeptide for import into the chloroplast. The transit sequence is proteolytically removed pnor to assembly ofsmall subunits with large subunits by chloroplast chaperonins (9). Synthesis of the large subunit in the chloroplast is also followed by posttranslational processing (16,25). The Nterminal Met-and Ser-2 are removed and Pro-3 acetylated in Rubisco from spinach (Spinacia oleracea L.), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), and muskmelon (Cucumis melo). Additionally, the tobacco and musk-'

No abstract

No abstract