Allosteric properties of the first enzyme of the histidine operon

PR-ATP synthetase, the first enzyme of histidine biosynthesis of Salmonella typhimurium has been purified by an improved procedure which yields enzyme which migrates as a single band in both gel electrophoresis and electrofocusing experiments. When stored in glycerol solution at −15°C, PR-ATP synthetase remains fully active and sensitive to inhibition by histidine for extended time periods. The enzyme requires manganese or magnesium ions for activity and is activated by numerous monovalent cations. The pH optimum is 8 to 10 and is strongly dependent upon the buffer employed. The equilibrium constant for the reaction is 10⁻³.     

Biochemical-genetic study of the first enzyme of histidine biosynthesis in Salmonella typhimurium: substrate and feedback binding regions.

Twenty-five strains of Salmonella typhimurium containing different mutations in the first gene of histidine biosynthesis were studied to correlate regions of the genetic map with biochemical functions. These strains contained either missense, double-frameshift, or suppressed nonsense mutations, all of which resulted in altered, though active, enzymes. Each mutant enzyme was assayed for activity in the presence of varying concentrations of the feedback inhibitor L-histidine or the substrates ATP and 5-phosphoribosyl-1-pyrophosphate. The feedback properties and substrate kinetics of each mutant enzyme were compared to wild-type values, and these results indicated that the following functions were correlated with regions of the hisG gene: feedback inhibition in two general areas, including regions IA and IB and regions V, VI, and VII; ATP binding in two general areas, including regions IA, IB, and II and regions V, VI, and VII; and 5-phosphoribosyl-1-pyrophosphate binding in two general areas, including regions IB, II, and III and regions V and VI.     

Derepression and repression of the histidine operon: role of the feedback site of the first enzyme.

Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAHis, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole.     

An operon for histidine biosynthesis in Streptomyces coelicolor

Summary In order to demonstrate that a cluster of five his genes (eight cistrons) on the circular chromosome of S. coelicolor is an operon, a constitutive mutant was characterized biochemically, and some aspects of enzyme repression were studied.The specific activities of three enzymes, two of which coded by genes of the his cluster and one specified by a his gene located far from the his cluster, were tested under different repression and derepression conditions and at various times of grwoth in a constitutive his mutant, in two leaky his mutants and in the wild type strains of S. coelicolor.The results of such investigations demonstrate that the constitutive mutant is derepressed exclusively for the synthesis of enzymes coded by genes of the his cluster; moreover only the synthesis of such enzymes takes place in a strictly coordinate way, suggesting that the his cluster behaves as a single unit of expression and regulation.     

Composition of the first enzyme of histidine biosynthesis isolated from wild-type and mutant operator strains of Salmonella typhimurium.

The first enzyme of histidine biosynthesis in Salmonella typhimurium, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), has been purified from two bacterial strains containing histidine operator deletions and compared to the eenzyme from a strain that has a normal operator. The enzymes isolated in different ways also are compared. Evidence as to the separateness of the operator and first structural gene or covalent modification of the first enzyme was sought. Specific activity, histidine feedback inhibition, amino acid analysis, discontinuous-gel electrophoresis, and gel filtration of the native enzyme, and Ouchterlony double-immunodiffusion tests were carried out. The purified enzyme contains little phosphorous and has five cysteine residues per subunit, which all are readily titratable. No evidence for differences in the enzyme preparations was obtained. Thus, no evidence for overlap of the histidine operator with the first structural gene was obtained.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.     

A sensitive assay for the reverse reaction of the first histidine biosynthetic enzyme.

A method capable of specifically detecting low levels of adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17) is given. It is based on conversion of the usual product of the enzyme, N¹-(5′-phospho-d-ribosyl)adenosine triphosphate to ATP, which is detected by luciferase luminescence utilizing a liquid scintillation spectrophotometer. The assay is linear in enzyme concentration down to a lower limit of at least 55 pg enzyme/0.2 ml.     

Biosynthetic direction substrate kinetics and product inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for the ATP phosphoribosyltransferase reaction in the biosynthetic direction were determined and are consistent with a sequential kinetic mechanism. To hold the fractions of magnesium-complexed substrates and products constant so as to avoid possible distortion of reciprocal velocity plots Mg²⁺ binding constants to the substrates ATP and phosphoribosylpyrophosphate and the product pyrophosphate were measured under assay conditions. Several conformational states of the phosphoribosyltransferase distinguishable by other criteria gave similar substrate kinetic behavior. Product inhibition studies were conducted to elucidate the binding order. Phosphoribosyl-ATP was competitive with respect to ATP and was non-competitive with respect to phosphoribosylpyrophosphate. Pyrophosphate was non-competitive with respect to both substrates. The data are consistent with the ordered Bi-Bi kinetic mechanism with ATP binding first to free enzyme and phosphoribosyl-ATP dissociating last from enzyme-product complexes.     

Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.

The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.     

An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis.

Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.     

The amino terminal sequence of ATP-phosphoribosyltransferase, the first gene product of the histidine operon.

The amino terminal sequence of ATP-phosphoribosyltransferase of $\text{Salmonella typhimurium}$ has been determined by automated Edman degradation. Since this protein is the first gene product of the histidine operon, comparison of its amino terminal sequence with the genetic code allows the deduction of a partial base sequence of its mRNA and the corresponding DNA. Five of the first nine residues of ATP-phosphoribosyltransferase align with the amino terminal sequence of histidinol dehydrogenase, the second gene product of the histidine operon.     

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.