Zone-to-zone projection of olfactory and vomeronasal sensory axons underlies the topographic and functional mapping of chemoreceptor expression zones of the sensory epithelia onto zonally arranged glomeruli in the main and accessory olfactory bulbs. Here we identified OCAM (R4B12 antigen), an axonal surface glycoprotein expressed by subsets of both olfactory and vomeronasal axons in a zone-specific manner. OCAM is a novel homophilic adhesion molecule belonging to the immunoglobulin superfamily with striking structural homology to neural cell adhesion molecule. In both the main and accessory olfactory systems, OCAM mRNA is expressed by sensory neurons in restricted chemoreceptor expression zones, and OCAM protein-expressing axons project to the glomeruli in the corresponding zones of the main and accessory bulbs. OCAM protein is expressed on subsets of growing sensory axons in explant cultures even in the absence of the target bulb. These results demonstrate a precisely coordinated zonal expression of chemoreceptors and OCAM and suggest that OCAM may play important roles in selective fasciculation and zone-to-zone projection of the primary olfactory axons.

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The wobble uridine in yeast cytosolic tRNA Lys2 UUU and tRNA Glu3UUC undergoes a thio-modification at the second position (s 2 modification) and a methoxycarbonylmethyl modification at the fifth position (mcm 5 modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s 2 modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitinlike protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s 2 modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s 2 modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s 2 and mcm 5 modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s 2 modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.Many modified nucleotides are found in tRNAs of various organisms, and the post-translational modification of tRNA molecules is thought to be necessary to maintain their structure and thereby to exert their proper function in translation (1). In yeast, uridine of the first position of anticodon, in cytosolic tRNA (cy-tRNA), 2 for lysine (cy-tRNA Lys2 UUU ) and glutamate (cy-tRNA Glu3 UUC ) contains sulfur instead of oxygen at the second position (the s 2 modification) and 5-methoxylcarbonylmethyl at the fifth position (the mcm 5 modification). These cytRNAs read split codon boxes; they decode the general NAAtype codon and can wobble into the NAG codon (2). Thus, the s 2 and mcm 5 modifications in U 34 are thought to be important in maintaining stable codon-anticodon pairing during decoding of these cy-tRNAs on the ribosome.For the s 2 modification of cy-tRNA Lys2 UUU and cytRNA Glu3 UUC , the cysteine desulfurase Nfs1 located in the mitochondria was essential, indicating that a sulfur atom used for the s 2 modification should originate from the cysteine sulfur atom located inside the mitochondria (3). Nfs1 is also known to provide sulfur for the iron-sulfur (Fe/S) cluster biosynthesis, which involves the mitochondrial ISC and cytosolic CIA machineries (4 -6). We previously demonstrated that the s 2 modification of cy-tRNAs was dependent not only on Nfs1 but also on other ISC and CIA proteins such as Cfd1 (7). Because the cytosolic Fe/S cluster assembly mediated by CIA must precede Fe/S cluster biosynthesis in the mitochondria, which is mediated by ISC (8), our previous observation suggests that at least one cytosolic Fe/S cluster-containing protein plays an indispensable role in the s 2 modification of cy-tRNAs (7). It may also be possible that the sulfur atom forming an Fe/S cluster may itself be directly used for the s 2 modification. Besides the mitochondrial Nfs1 and Fe/S cluster assembly...

Pyridoxal 5'-phosphate (PLP) acts as the coenzyme in a vast number of reactions in amino acid metabolism. The study of PLP enzymes is one of the most fascinating frontiers in enzymology, and now the mechanism s of several types of PLP enzymes are being discussed at the atomic level based on crystallographic, spectroscopic, and site-directed mutagenesis studies. In this review, I summarize the important findings, including those provided by classical studies, on the reaction mechanisms of several PLP enzymes, with the intention of discussing the chemically and thermodynamically consistent principle of the catalytic action of PLP enzymes common to all the enzymes of this group, and the uniqueness of individual enzymes that endows them substrate and reaction specificity.

; otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys 39 and Tyr 265. The results of molecular orbital calculation also support this mechanism.