The Salmonella typhimurium glycine cleavage enzyme system

Summary A glycine cleavage enzyme system, inducible by glycine, has been demonstrated in Salmonella typhimurium. The induced enzyme levels, however, are only about 20% of the induced levels found in Escherichia coli. Starting with a serine auxotroph, mutants were isolated that grow with a serine supplement, but not with a glycine supplement. Three independently isolated mutants have reduced or nondetectable glycine cleavage enzyme levels. The new mutations, designated gcv, were mapped between the serA and lys genes at 62.5 min on the S. typhimurium chromosome.Abbreviations C1 one-carbon - GCV glycine cleavage - GM glucose minimal - L agar Luria agar - LB Luria broth - Tc tetracycline     

Specificity of the cholesterol side-chain cleavage enzyme system of adrenal cortex

Incubation of lanosta-8, 24-dien-3β-o1-1,2- ³H and lanost-8-en-3β-o1-1, 2-³H with an adrenocortical bovine mitochondrial acetone-dried preparation did not yield any significant ( < 0.01%) 3β-hydroxy-4, 4, 14-trimethyl-5α-pregn-8-en-20-one. Under the same conditions cholesterol-1,2-³H yielded 8.3% pregnenolone. Incubation of (20S?) — 17α, 20-dihydroxycholesterol-7-³ H yielded 0.6 to 1.6% (20SS?, 22R?) — 17α, 20, 22-trihydroxycholesterol, 1.0 to 3.2% of 17α-hydroxypregnenolone, but no significant ( < 0.02%) (20S, 22S)-17α, 20, 22-trihydroxycholesterol. In another experiment incubation of cholesterol-1, 2-³H yielded 5% pregnenolone, 0.5% 17α-hydroxypregnenolone, 0.2% (20R?,22R?)-20, 22-dihydroxy-cholesterol, but no significant ( < 0.01%) 17α-hydroxy-cholesterol, (20S?) -17α, 20-dihydroxycholesterol or (20S?, 22R?)-17α, 20, 22-trihydroxycholesterol.     

Positive regulation of the Escherichia coli glycine cleavage enzyme system.

A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv.     

The mitochondrial glycine cleavage system

Summary The glycine cleavage enzyme system is composed of four different proteins tentatively called P-protein, H-protein, T-protein and L-protein, and catalyzes the following reaction reversibly: Glycine + tetrahydrofolate + NAD⁺ 5, 10-methylene-tetrahydrofolate + NH₃ + CO₂ + NADH + H^– Glycine decarboxylase, tentatively called P-protein, is able by itself to catalyze glycine decarboxylation, yielding methylamine as product, but at an extremely low rate. P-Protein alone is also able to catalyze slightly the exchange of carboxyl carbon of glycine with CO₂. However, the rates of the P-protein-catalyzed reactions are greatly increased by the co-existence of aminomethyl carrier protein, a lipoic acid-containing enzyme tentatively called H-protein. Several lines of evidence suggest that H-protein brings about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein and that the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-Protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine. The idea that H-protein functions as a modulator of P-protein was further supported by the study of a patient with nonketotic hyperglycinemia. The primary lesion in this patient appeared to consist in structural abnormality in H-protein; the H-protein purified from the liver of this patient was apparently devoid of functional lipoic acid. Nevertheless, H-protein from the patient could stimulate the P-protein-catalyzed exchange of the carboxyl carbon of glycine and CO₂, although only to a limited extent. The observed activity should be independent of the functioning of lipoic acid and would be a reflection of a conformational change in P-protein brought about by H-protein.P-Protein was inactivated when it was incubated with glycine in the presence of II-protein, and the inactivation was completely prevented when bicarbonate was further added so as to allow the glycine-CO₂ exchange to proceed. The inactivation was accompanied by a spectral change of P-protein. The inactivation of P-protein seemed to take place as a side reaction of the glycine decarboxylation and to reflect the formation of a ternary complex of P-protein, H-protein and aminomethyl moiety of glycine through a Schiff base linkage of the H-protein-bound aminomethyl moiety with the pyridoxal phosphate of P-protein.     

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Restriction enzyme cleavage of ultraviolet-damaged DNA

SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.     

Photon enzyme activation

The electromagnetic molecular electronic resonance (EMER) frequencies of the molecular chains of α-chymotrypsin are calculated. The chain length relations and coupling positions suggest a possible energy transfer, at the EMER frequencies, from one chain to the other. Photon enzyme activation data indicate that the energy corresponding to the EMER frequencies of its molecular chains is used by α-chymotrypsin for its enzyme function.