J.E. Folk

Transglutaminase: Mechanistic features of the active site as determined by kinetic and inhibitor studies

Abstract 1. 1. The K m values for the transglutaminase-catalyzed (amine: R-glutamine transferase (hydrolyzing) hydrolysis and hydroxylamine incorporation reactions and the K n values for inhibition by carbobenzoxy-(CBZ)-amino acids and CBZ-peptides, all obtained as functions of pH, reveal an enzyme group of pK 7.5 to 8.5. Inhibitor specificity studies indicate that this group is involved in binding substrate at the peptide bond formed through the α-amino group of the glutamine residue. 2. 2. Inhibitor constants for chloroacetyl amino acids show no variation with pH. It is suggested that these inhibitors, as well as chloroacetic acid, are bound to an enzyme active center group through the carbonyl carbon of their chloroacetyl position. The change in K i values for chloroacetic acid at about pH 6.5 has been attributed to an induced shift in pK of another group which is in the close structural vicinity. 3. 3. This group, tentatively identified as an-SH on the basis of its sensitivity to thiol reagents, has an apparent pK of around 6.5, as judged from activity titration versus p H with chloroacetamide, decrease in v max . with pH and induced shift in its pK with chloroacetic acid. 4. 4. The efficient protection against thiol reagent inhibition by substrate supports a supposition that this -SH group forms a covalent bond with substrate during the catalytic reaction.

Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation.

The formation of the unusual amino-acid hypusine in eIF-5A (eukaryotic initiation factor 5A) is associated with cellular proliferation. We used a panel of compounds, including mimosine, to probe the relationship between the exit from the G1 phase of the cell cycle, i.e., the onset of DNA replication, and the formation of hypusine by the enzyme deoxyhypusyl hydroxylase (DOHH). These two parameters displayed the same dose dependency and structure-activity relationship. Only compounds that inhibited DOHH also suppressed proliferation. This effect was observed: (i) in spontaneously proliferating, virally transformed, and mitogen-stimulated cells; (ii) for both anchorage-dependent and anchorage-independent proliferation; and (iii) with normal and malignant cell lines. DOHH reactivation occurred rapidly after inhibitor withdrawal and correlated with synchronized entry into S. The changes in the expression of specific genes during the G1-to-S transition mimicked the physiological pattern. These findings suggest that hypusine formation in eIF-5A which occurs in a specific, invariant sequence motif acquired early in evolution, may be involved in the G1-to-S transition in the eukaryotic cells tested.

Influence of cobalt and cadmium on the peptidase and esterase activities of carboxypeptidase B

1. 1. A 100% increase in the peptidase activity of carboxypeptidase B has been observed as a result of incubation of this enzyme with cobaltous ions. An apparent reduction in esterase activity accompanies this peptidase-activating effect under certain experimental conditions. 2. 2. Incubation of carboxypeptidase B with Cd++, on the other hand, results in as much as 100% increase in esterase activity, attended by a parallel loss in peptidase activity. 3. 3. Incubation with Co++ or Cd++ results in a reduction in the zinc content of the enzyme and a concomitant binding of the metal ion employed in incubation. 4. 4. Treatment of the cobalt and cadmium carboxypeptidases B with zinc essentially restores the peptidase and esterase activities to the levels of those of the untreated purified enzyme. 5. 5. The results support the hypotheses that the mechanism of catalytic action of carboxypeptidases A and B are similar and that the catalytic properties of carboxypeptidase B are a function of the metal bound to the enzyme protein at the level of 1 g atom/mole.

Chymotrypsin C: III. Sequence of amino acids around an essential histidine residue

Abstract Evidence is presented that inhibition of chymotrypsin C by tosyl- l -leucine chloromethylketone results from the reaction of this reagent with a single essential histidine residue of the enzyme. Labeled peptides containing this histidine residue have been isolated from peptic digests of [14C]tosyl- l -leucine chloromethylketone-inhibited chymotrypsin C. Analysis of these peptides supports a postulated sequence of amino acids surrounding this essential histidine residue of Ala-Ala-His-Cys-Ile-Asp Ser-Gly-Thr-Ser-Arg-Thr.

Inhibition of lysine and lysine peptide utilization in bacteria by peptides of S-(β-aminoethyl)-cysteine☆

Abstract 1. 1. The preparation and properties of L -thiosylglycine [S-(β-aminoethyl)- L -cysteinyl-glycine] and glycyl- L -thiosine[glycyl-S-(β-aminoethyl)- L -cysteine] and several other thiosine derivatives are reported. 2. 2. The effects of these thiosine peptides as inhibitors of lysine utilization in bacteria has been compared to those previously reported for thiosine. 3. 3. Reversal of thiosine peptide growth inhibition by lysine and peptides of lysine in two strains of microorganisms has been studied.