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The reaction pyruvate + CoA % acetyl-CoA + formate, catalyzed by pyruvate formate-lyase of Escherichia coli, occurs by the succeeding half-reactions (a) E + pyruvate e E-acetyl + formate; (b) E-acetyl + CoA Making use of coupled optical assays, a 'ping-pong mechanism' was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K = 750 (30 "C, pH 8.1), which agrees with chemically determined values.The intermediate acetyl-enzyme, which had been previously proposed from the ['4C]formatepyruvate exchange, was detected by product-pulse experiments with [2-'4C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein.The value of the equilibrium constant of the first half-reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl-enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl-CoA.The intermediate was found to react with dithiothreitol with a second-order rate constant at 30°C and pH 7.6 of 1160 M-' xmin-'. It resulted in a half-life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization.The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme-11-dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S-adenosylmethionine and oxamate (as a pyruvate analogue).The conversion of pyruvate into acetyl-CoA according to the equation Pyruvate + CoASH e Acetyl-SCoA + formate (1) plays a key role in the metabolism of Escherichia coli. It is the anaerobic counterpart of pyruvate oxidation by the pyruvate dehydrogenase complex and is operative alternatively to the latter [1,2].Being a unique type of reaction of a 2-oxocarbonic acid, the mechanism of the pyruvate formate-lyase Abbreviation. Mops, 2-(N-morpholino)propanesulfonate. Enzymes (CBN recommendations 1972). Pyruvate for-

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaLl, MvaL10, and MvaL12, encoded by the MvaLl operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaLl, the homolog of the regulatory protein Li encoded by the Lll operon of Escherichia coli, was shown to be an autoregulator of the MvaLl operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaLl was localized by site-directed mutagenesis to a region within the coding sequence of the MvaLl gene commencing about 30 bases downstream of the ATG initiation codon. The MvaLl binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the Li regulatory target site ofE. coli and to the putative binding site for MvaLl on the 23S rRNA. In contrast to other regulatory systems, the putative MvaLl binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.Ribosomes from different species of archaebacteria have been investigated extensively in the past, largely because they have proven to be excellent phylogenetic probes to study molecular evolution (38,39). A large amount of data is now available on ribosomal (r) proteins of archaebacteria. On the whole, the primary structures of archaebacterial r proteins are more related to their eukaryotic than to their eubacterial counterparts. In contrast, the genes for r proteins are arranged in operons as in eubacteria; even the order of the genes within the operons is similar to that found in eubacteria. Methanococcus vannielii, a methanogenic archaebacterium, contains more than 60 r proteins (49), a value intermediate between that found in eubacteria and that in eukaryotes. In general, the Methanococcus r proteins have higher molecular masses than their eubacterial homologs. As in Escherichia coli, the archaebacterial r protein equivalent to L12 is present in four copies per ribosome (7). In M. vannielii, three r protein operons have been characterized in detail: two transcription units corresponding to the E. coli spectinomycin and streptomycin operon (1,35), and the MvaLl operon (3). The MvaLl operon encodes the r proteins MvaLi, MvaL10, and MvaL12 (Fig. 1) rium cutirubrum (50) and in the sulfur-dependent thermophilic archaebacterium Sulfolobus solfataricus (51), the equivalent r protein genes are clustered in the same order as in E. coli. The regulation of the expression of many r protein operons has been investigated in E. coli (27,43). In most cases, a translational feedback regulation exists, in which certain r proteins act as autogenous repressors, controlling the expression of the operon in which they...