Kinetics of formation and dissociation of metallocarboxypeptidases.

The rates of formation of a number of metallocarboxypeptidases from metal ions and bovine apocarboxypeptidase A (CPA) have been measured directly and by a competitive method. Rates were determined with pH = 6-8 by utilising the pH change attending metal-ion incorporation, employing indicator and stopped-flow. Second-order rate constants Kf, M-1 s-1 at 25 degrees C, I = 1 M NaCl, pH = 7, Tris = 25 micrometer) were 1.7 X 10(5) (Mn2+), 3 X 10(4) (Co2+), 5 X 10(3) (Ni2+), 7 X 10(5) Zn2+), and 9 X 10(5) (Cd2+). Relative incorporation rate constants were determined at 25 degrees, pH = 7.0, Tris = 0.1 M, by competing two metal ions for a deficiency of apoprotein and analyzing the products by differential enzyme activity. Agreement between the two methods was reasonable. Rate constants for dissociation of CoCPA, NiCPA, and ZnCPA were measured by loss of enzyme activity on addition of the metal ion scavenger EDTA. Values of kd at 25 degrees, I = 1.0 M NaCl, pH = 7.0 were 8 X 10(-3), 3 X 10(-5), and 4 X 10(-4) s(-1), respectively. Values of K obtained kinetically (kf/kd) were in good agreement with those determined by activity measurements of equilibrated solutions. Results are compared with those of bovine apocarbonic anhydrase, where generally significantly slower rates are encountered.     

Progress in metallocarboxypeptidases and their small molecular weight inhibitors

In what corresponds to a life span, metallocarboxypeptidases (MCPs) have jumped from being mere contaminants in animal pancreas powders (in depression year 1929) to be key players in cellular and molecular processes (in yet-another-depression years 2009–2010). MCPs are unique zinc-dependent enzymes that catalyze the breakdown of the amide bond at the C-terminus of peptide and protein substrates and participate in the recovery of dietary amino acids, tissue organogenesis, neurohormone and cytokine maturation and other important physiological processes. More than 26 genes code for MCPs in the human genome, many of them still waiting to be fully understood in terms of physiological function. A variety of MCPs have been linked to diseases in man: acute pancreatitis and pancreas cancer, type 2 diabetes, Alzheimer’s Disease, various types of cancer, and fibrinolysis and inflammation. Many of these discoveries have been made possible thanks to recent advances, as exemplified by plasma carboxypeptidases N and B, known for fifty and twenty years, respectively, which have had their structures released only very recently. Plasma carboxypeptidase B is a biological target for therapy because of its involvement in the coagulation/fibrinolysis processes. Besides, the widespread use of carboxypeptidase A as a benchmark metalloprotease since the early days of Biochemistry has allowed the identification and design of an increasingly vast repertory of small molecular weight inhibitors. With these two examples we wish to emphasize that MCPs have become part of the drug discovery portfolio of pharmaceutical companies and academic research laboratories. This paper will review key developments in the discovery and design of MCP small molecular weight inhibitors, with an emphasis on the discovery of chemically diverse entities. Although encouraging advances have been achieved in the last few years, the specificity and oral bioavailability of the new chemotherapeutic agents seem to pose a challenge to medicinal chemists.     

Structure and mechanism of imidazoleglycerol-phosphate dehydratase

The structure of A. thaliana imidazoleglycerol-phosphate dehydratase, an enzyme of histidine biosynthesis and a target for the triazole phosphonate herbicides, has been determined to 3.0 A resolution. The structure is composed of 24 identical subunits arranged in 432 symmetry and shows how the formation of a novel dimanganese cluster is crucial to the assembly of the active 24-mer from an inactive trimeric precursor and to the formation of the active site of the enzyme. Molecular modeling suggests that the substrate is bound to the manganese cluster as an imidazolate moiety that subsequently collapses to yield a diazafulvene intermediate. The mode of imidazolate recognition exploits pseudosymmetry at the active site arising from a combination of the assembly of the particle and the pseudosymmetry present in each subunit as a result of gene duplication. This provides an intriguing example of the role of evolution in the design of Nature's catalysts.     

Structure and mechanism of O-acetylserine sulfhydrylase

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.     

Structure and functioning mechanism of transketolase

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.     

Structure and mechanism of spermidine synthases

Aminopropyltransferases transfer aminopropyl groups from decarboxylated S-adenosylmethionine to amine acceptors, forming polyamines. Structural and biochemical studies have been carried out with the human spermidine synthase, which is highly specific for putrescine as the amine acceptor, and the Thermotoga maritima spermidine synthase, which prefers putrescine but is more tolerant of other substrates. Comparison of the structures of the human spermidine synthase with both substrates and products with the known structure of T. maritima spermidine synthase complexed to a multisubstrate analogue inhibitor and analysis of the properties of site-directed mutants provide a general mechanistic hypothesis for the aminopropyl transfer reaction. The studies also provide a structural basis for the specificity of the spermidine synthase subclass of the aminopropyltransferase family.     

Structure and mechanism of intramembrane protease

Many functionally important membrane proteins are cleaved within their transmembrane helices to become activated. This unusual reaction is catalyzed by a group of highly specialized and membrane-bound proteases. Here I briefly summarize current knowledge about their structure and mechanism, with a focus on the rhomboid family. It has now become clear that rhomboid protease can cleave substrates not only within transmembrane domains, but also in the solvent-exposed juxtamembrane region. This dual specificity seems possible because the protease active site is positioned in a shallow pocket that can directly open to aqueous solution through the movement of a flexible capping loop. The narrow membrane-spanning region of the protease suggests a possible mechanism for accessing scissile bonds that are located near the end of substrate transmembrane helices. Similar principles may apply to the metalloprotease family, where a crystal structure has also become available. Although how the GxGD proteases work is still less clear, recent results indicate that presenilin also appears to clip substrate from the end of transmembrane helices.     

Structure and mechanism of RNA ligase

T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing ligases (RELs) of Trypanosoma and Leishmania. The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3'-OH and 5'-PO4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA ligases, DNA ligases, and mRNA capping enzymes.     

Structure and mechanism of iron-only hydrogenases

The recent elucidation of the structures of iron-only hydrogenases from the microorganisms Clostridium pasteurianum and Desulfovibrio desulfuricans has revealed that the presumed site of reversible hydrogen oxidation exists as a unique, protein-associated organometallic prosthetic group. Details of the hydrogenase structures provide insight into the chemical mechanism of this highly evolved catalyst.     

Structure and mechanism of purine-binding riboswitches

A riboswitch is a non-protein coding sequence capable of directly binding a small molecule effector without the assistance of accessory proteins to regulate expression of the mRNA in which it is embedded. Currently, over 20 different classes of riboswitches have been validated in bacteria with the promise of many more to come, making them an important means of regulating the genome in the bacterial kingdom. Strikingly, half of the known riboswitches recognize effector compounds that contain a purine or related moiety. In the last decade, significant progress has been made to determine how riboswitches specifically recognize these compounds against the background of many other similar cellular metabolites and transduce this signal into a regulatory response. Of the known riboswitches, the purine family containing guanine, adenine and 2'-deoxyguanosine-binding classes are the most extensively studied, serving as a simple and useful paradigm for understanding how these regulatory RNAs function. This review provides a comprehensive summary of the current state of knowledge regarding the structure and mechanism of these riboswitches, as well as insights into how they might be exploited as therapeutic targets and novel biosensors.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B(12) importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored.     

Structure and mechanism of tryptophylquinone enzymes

Tryptophylquinone cofactors are formed by posttranslational modifications that result in the incorporation of two oxygens into a tryptophan side chain, and the covalent cross-linking of that side chain to another amino acid residue. Tryptophylquinone enzymes catalyze the oxidative deamination of primary amines, and utilize other redox proteins as electron acceptors. Mechanistic and structural studies of these enzymes are providing insight into how these enzymes utilize these highly reactive protein-derived quinones in a controlled manner to facilitate biologically important catalytic and electron transfer reactions.     

Structure and mechanism of proton-translocating transhydrogenase

Recent developments have led to advances in our understanding of the structure and mechanism of action of proton-translocating (or AB) transhydrogenase. There is (a) a high-resolution crystal structure, and an NMR structure, of the NADP(H)-binding component (dIII), (b) a homology-based model of the NAD(H)-binding component (dI) and (c) an emerging consensus on the position of the transmembrane helices (in dII). The crystal structure of dIII, in particular, provides new insights into the mechanism by which the energy released in proton translocation across the membrane is coupled to changes in the binding affinities of NADP(+) and NADPH that drive the chemical reaction.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters are central to many physiological processes, including the uptake of nutrients, the non-classical secretion of signaling molecules and toxins, multidrug resistance and the development of human disease. As one might expect from this spectrum of translocation events, these ubiquitous, ATP-dependent pumps or channels are capable of transporting an enormous variety of substrates, ranging from small ions to large proteins. Recently determined structures of full-length ABC transporters and isolated ABC domains have increased our understanding of the functional mechanism of these proteins.