Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural databases.

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(Hydroxymethy1)bilane synthase (HMBS) catalyses the conversion of porphobilinogen (2) into the (hydroxymethy1)bilane derivative 3, a linear tetrapyrrolic intermediate in the biosynthesis of haem, chlorophyll, and related pigments. The conversion involves the sequential formation of four intermediate covalent enzyme-substrate complexes, before the product is released. We analysed the pre-steady-state kinetics of the formation of the complexes, taking advantage of their remarkable chemical stability allowing chromatographic separation. The experimental approach involved the generation of the complexes while HMBS was immobilised on an anion-exchange column. A solution being 0.2 K,,, in substrate was pumped through the column during a time interval which was varied to sample the pre-steady-state period. Then, the enzyme and enzyme-substrate complexes were eluted and their proportions evaluated. A computer simulation of the pre-steady-state time course, in combination with a x 2 fitting to the experimental data, allowed the specificity constants k,,JK, for the individual steps of the process to be derived. By repeating the analysis with variants of HMBS in which specific amino acids were replaced by others, we demonstrated that it is possible to trace the consequences of amino-acid replacements down to the individual steps of the reaction sequence. Since the positions of the amino acids concerned in the three-dimensional structure were known, detailed structure-function relationships become evident in this way.Introduction. -(Hydroxymethy1)bilane synthase (HMBS, EC 4.3.1.8, also known as porphobilinogen deaminase) is an enzyme of the biosynthetic pathway leading to haem, chlorophyll, vitamin B,,, coenzyme F430, and related tetrapyrrolic pigments. It catalyses the conversion of four equivalents of porphobilinogen (PBG; 2) into (hydroxymethy1)-bilane derivative 3 and ammonia [ 11 [2] (Scheme I ) .HMBS is an enzyme converting PBG into products obeying Michaelis-Menten kinetics (k,,, = 0.1 s-', K,,, = 5-10 ~L M at pH 7.4 for HMBS from Escherichia coli [3]) [4] and displaying chemical reactivity of a polymerase [S]. The signal to stop polymerisation does not require any external factors, but is built into the HMBS molecule itself as soon as four substrate molecules have been processed, the tetrameric product is released. To accomplish the assembly of the bilane, HMBS uses a unique dipyrrin ( = dipyrromethane) cofactor to which the growing chain remains covalently attached [6] [7] (see Scheme 2). The cofactor is in turn covalently bound to the enzyme via the S-atom of a cysteine residue (Cys-242 of the enzyme from E.coli). The HMBS apoenzyme, lacking the cofactor, is capable of assembling its own cofactor from two substrate molecules. Being derived from PBG (2), the cofactor can be isotopically labelled by

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A procedure was developed for the biosynthetic preparation of "N-labelled guanosine and inosine through the action of a mutant Bacillus subtilis strain. Crude [N', 1,3,7,guanosine and [1,3,7,9-''N]inosine were isolated from the culture filtrate by precipitation and anion-exchange chromatography (Scheme 1 ). No cell lysis and no enzymatic degradation was necessary. The per-isobutyrylated derivatives 1 and 2 were isolated from a complex mixture, purified by virtue of their different lipophilicity, and separated in three steps involving normaland reversed-phase silica-gel chromatography. One litre of complex nutrient medium yielded 8.44 mmol of guanosine derivative and 2.84 mmol of inosine derivative with high average ''N enrichment (83.5 and 91.9 atom-%, resp.). [N6,1,3,7,9-"N]Adenosine (4) was obtained from 2',3',5'-tri-O-is0butyryl[l,3,7,9-'~N]inosine (1) through the ammonolysis of its 1,2,4-triazolyl derivative with aqueous "NH, (Scheme 2).Introduction. -The advent of multidimensional heteronuclear 'H-NMR spectroscopy for the elucidation of the solution structure of large biomolecules [l] [2] led to a number of investigations aiming at the preparation of defined "N-labelled RNA fragments [3] [4]. The general approach was a biosynthetic one: Microorganisms grown on minimal media containing ['5NJammonium salts were used to produce labelled RNA. After enzymatic degradation to the nucleosides and enzymic phosporylation, all four "N-labelled ribonucleoside 5'-triphosphates were obtained in 1-100 pmol amounts per litre of culture medium. Those were used as substrates for an enzymic in vitro RNA synthesis by T7 RNA polymerase.Our approach divides the preparation of "N-labelled ribonucleosides into a biosynthetic and a synthetic one, with the aim to obtain g-quantities of the nucleosides for their use in both in vitro and chemical [U-I5N]RNA synthesis. Here, we describe the preparation of the purine nucleosides [ l,3,7,9-'5N]inosine and [N2, 1 ,3,7,9-'SN]guanosine. They were directly obtained from a conventionally cultivated mutant Bacillus subtilis strain NA6601 [5]. Under certain growth conditions, these Gram -positive bacteria are capable of releasing large quantities of guanosine (G) and inosine (I) into the growth medium. This greatly simplifies the isolation and purification procedure. Subsequently, [ 1,3,7,9-''PJlinosine was chemically converted into [N6, 1,3,7,9-"N]adenosine. Results andDiscussion. -Production, Isolation, and Purification of the Main Metabolites. Optimal aerobic growth of B. subtilis NA6601 required a medium based on sorbitol and autolyzed yeast (SD medium) for precultures and a complex medium based on I) Diploma thesis ( A . N . 1991, M . M . and T.E. 1992, 0. B. 1993).