Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria

Peptide inhibitors of Streptomyces DD-carboxypeptidases

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms

Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.

Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

The peptidoglycan crosslinking enzyme system in Streptomyces R61, K15 and rimosus. Immunological studies

The exocellular DD-carboxypeptidases from Streptomyces R61, K 15, the lysozyme-releasable DD-carboxypeptidases from Streptomyces R61, K15 and rimosus, and the membrane-bound DD-carboxypeptidase of Streptomyces K15 are immunologically related to each other

The penicillin receptor in Streptomyces

Kinetics and optical studies of Streptomyces DD-carboxypeptidases-transpeptidases led to the conclusion that the donor, acceptor, and penicillin sites on these enzymes are different but not independent and that penicillin acts as a modifier of the conformation of the protein. In the presence of penicillin, the penicillin-sensitive enzymes would be frozen in a conformation that prevents catalytic activity

On the DD-carboxypeptidase enzyme system of Streptomyces strain K15

Streptomyces K15 possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains on single penicillin-binding protein. The exocellular 54000-Mr enzyme is extremely sensitive to benzylpenicillin and performs low transpeptidase activity on the carbonyl-donor/amino-acceptor tetrapeptide ACLLys(Gly)-DAla-DAla. The exocellular 40 000-Mr enzyme and the two lysozyme-releasable 40 000-Mr and 38 000-Mr enzymes are moderately sensitive to benzylpenicillin and have a high propensity to catalyse dimer formation from the aforementioned tetrapeptide monomer

Biochemistry of the Bacterial Wall Peptidoglycan in Relation to the Membrane

The peptidoglycan physically protects the membrane against deleterious influences. The membrane is involved in peptidoglycan synthesis and in other wall regulation mechanisms. The possible physiological function of a soluble carboxy-peptidase active on acyl-D-alanyl-D-R-OH substrates and its relation to the membrane-bound transpeptidase involved in peptidoglycan synthesis are discussed