Annual Review of Biochemistry - Volume 70, 2001

DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by nucleosomal barriers imposed by histone proteins. Among the well-known covalent modifications of histones, the reversible acetylation of internal lysine residues in histone amino-terminal domains has long been positively linked to transcriptional activation. Recent biochemical and genetic studies have identified several large, multisubunit enzyme complexes responsible for bringing about the targeted acetylation of histones and other factors. This review discusses our current understanding of histone acetyltransferases (HATs) or acetyltransferases (ATs): their discovery, substrate specificity, catalytic mechanism, regulation, and functional links to transcription, as well as to other chromatin-modifying activities. Recent studies underscore unexpected connections to both cellular regulatory processes underlying normal development and differentiation, as well as abnormal processes that lead to oncogenesis. Although the functions of HATs and the mechanisms by which they are regulated are only beginning to be understood, these fundamental processes are likely to have far-reaching implications for human biology and disease.

Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5′-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-β-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5′-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4 Fe–4S]⁺ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5′-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4 Fe–4S] proteins that catalyze the insertion of sulfur into unactivated C–H bonds, which are cleaved by the 5′-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5′-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites.

The three-dimensional structures of tryptophan synthase, carbamoyl phosphate synthetase, glutamine phosphoribosylpyrophosphate amidotransferase, and asparagine synthetase have revealed the relative locations of multiple active sites within these proteins. In all of these polyfunctional enzymes, a product formed from the catalytic reaction at one active site is a substrate for an enzymatic reaction at a distal active site. Reaction intermediates are translocated from one active site to the next through the participation of an intermolecular tunnel. The tunnel in tryptophan synthase is ∼25 Å in length, whereas the tunnel in carbamoyl phosphate synthetase is nearly 100 Å long. Kinetic studies have demonstrated that the individual reactions are coordinated through allosteric coupling of one active site with another. The participation of these molecular tunnels is thought to protect reactive intermediates from coming in contact with the external medium.

The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

The protein sequence and structure databases are now sufficiently representative that strategies nature uses to evolve new catalytic functions can be identified. Groups of divergently related enzymes whose members catalyze different reactions but share a common partial reaction, intermediate, or transition state (mechanistically diverse superfamilies) have been discovered, including the enolase, amidohydrolase, thiyl radical, crotonase, vicinal-oxygen-chelate, and Fe-dependent oxidase superfamilies. Other groups of divergently related enzymes whose members catalyze different overall reactions that do not share a common mechanistic strategy (functionally distinct suprafamilies) have also been identified: (a) functionally distinct suprafamilies whose members catalyze successive transformations in the tryptophan and histidine biosynthetic pathways and (b) functionally distinct suprafamilies whose members catalyze different reactions in different metabolic pathways. An understanding of the structural bases for the catalytic diversity observed in super- and suprafamilies may provide the basis for discovering the functions of proteins and enzymes in new genomes as well as provide guidance for in vitro evolution/engineering of new enzymes.

Protein tyrosine phosphatases (PTPs) are a diverse group of enzymes that contain a highly conserved active site motif, Cys-x5-Arg (Cx5R). The PTP super-family enzymes, which include tyrosine-specific, dual specificity, low-molecular-weight, and Cdc25 phosphatases, are key mediators of a wide variety of cellular processes, including growth, metabolism, differentiation, motility, and programmed cell death. The PTEN/MMAC1/TEP1 gene was originally identified as a candidate tumor suppressor gene located on human chromosome 10q23; it encodes a protein with sequence similarity to PTPs and tensin. Recent studies have demonstrated that PTEN plays an essential role in regulating signaling pathways involved in cell growth and apoptosis, and mutations in the PTEN gene are now known to cause tumorigenesis in a number of human tissues. In addition, germ line mutations in the PTEN gene also play a major role in the development of Cowden and Bannayan-Zonana syndromes, in which patients often suffer from increased risk of breast and thyroid cancers. Biochemical studies of the PTEN phosphatase have revealed a molecular mechanism by which tumorigenesis may be caused in individuals with PTEN mutations. Unlike most members of the PTP superfamily, PTEN utilizes the phosphoinositide second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3), as its physiologic substrate. This inositol lipid is an important regulator of cell growth and survival signaling through the Ser/Thr protein kinases PDK1 and Akt. By specifically dephosphorylating the D3 position of PIP3, the PTEN tumor suppressor functions as a negative regulator of signaling processes downstream of this lipid second messenger. Mutations that impair PTEN function result in a marked increase in cellular levels of PIP3 and constitutive activation of Akt survival signaling pathways, leading to inhibition of apoptosis, hyperplasia, and tumor formation. Certain structural features of PTEN contribute to its specificity for PIP3, as well as its role(s) in regulating cellular proliferation and apoptosis. Recently, myotubularin, a second PTP superfamily enzyme associated with human disease, has also been shown to utilize a phosphoinositide as its physiologic substrate.

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (β, γ, δ, and ∍), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [PtdIns(3)P, βγ subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound α subunit of Gq) of PLC-β; the PH domain [PtdIns(3,4,5)P3] and Src homology 2 domain [tyrosine-phosphorylated proteins, PtdIns(3,4,5)P3] of PLC-γ; the PH domain [PtdIns(4,5)P2] and C2 domain (Ca²⁺) of PLC-δ; and the Ras binding domain (GTP-bound Ras) of PLC-∍. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.

Cys2His2 zinc finger proteins offer a stable and versatile framework for the design of proteins that recognize desired target sites on double-stranded DNA. Individual fingers from these proteins have a simple ββα structure that folds around a central zinc ion, and tandem sets of fingers can contact neighboring subsites of 3–4 base pairs along the major groove of the DNA. Although there is no simple, general code for zinc finger–DNA recognition, selection strategies have been developed that allow these proteins to be targeted to almost any desired site on double-stranded DNA. The affinity and specificity of these new proteins can also be improved by linking more fingers together or by designing proteins that bind as dimers and thus recognize an extended site. These new proteins can then be modified by adding other domains—for activation or repression of transcription, for DNA cleavage, or for other activities. Such designer transcription factors and other new proteins will have important applications in biomedical research and in gene therapy.

The nuclear peroxisome proliferator–activated receptor γ (PPARγ) is a transcription factor that is activated by polyunsaturated fatty acids and their metabolites and is essential for fat cell formation. Although obesity is a strong risk factor for type 2 diabetes mellitus and other metabolic diseases, potent PPARγ activators such as the glitazone drugs lower glucose and lipid levels in patients with type 2 diabetes and also have antiatherosclerotic and antihypertensive effects. We review recent studies providing insight into the paradoxical relationship between PPARγ and metabolic disease. We also review recent advances in understanding the structural basis for PPARγ activation by ligands. The unusual ligand-binding properties of PPARγ suggest that it will be possible to discover new chemical classes of receptor “modulators” with distinct pharmacological activities for the treatment of type 2 diabetes and other metabolic diseases.

DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement. For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.

The ribosome discriminates between correct and incorrect aminoacyl-tRNAs (aa-tRNAs), or their complexes with elongation factor Tu (EF-Tu) and GTP, according to the match between anticodon and mRNA codon in the A site. Selection takes place at two stages, prior to GTP hydrolysis (initial selection) and after GTP hydrolysis but before peptide bond formation (proofreading). In part, discrimination results from different rejection rates that are due to different stabilities of the respective codon-anticodon complexes. An important additional contribution is provided by induced fit, in that only correct codon recognition leads to acceleration of rate-limiting rearrangements that precede chemical steps. Recent elucidation of ribosome structures and mutational analyses suggest which residues of the decoding center may be involved in signaling formation of the correct codon-anticodon duplex to the functional centers of the ribosome. In utilizing induced fit for substrate discrimination, the ribosome resembles other nucleic acid–programmed polymerases.

A decade after the discovery of electrospray and matrix-assisted laser desorption ionization (MALDI), methods that finally allowed gentle ionization of large biomolecules, mass spectrometry has become a powerful tool in protein analysis and the key technology in the emerging field of proteomics. The success of mass spectrometry is driven both by innovative instrumentation designs, especially those operating on the time-of-flight or ion-trapping principles, and by large-scale biochemical strategies, which use mass spectrometry to detect the isolated proteins. Any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry. As has already happened in genomics, increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data. Protein-protein interactions can be analyzed directly by precipitation of a tagged bait followed by mass spectrometric identification of its binding partners. By these and similar strategies, entire protein complexes, signaling pathways, and whole organelles are being characterized. Posttranslational modifications remain difficult to analyze but are starting to yield to generic strategies.

The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity. A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin. A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action. Coactivator functions can be broadly divided into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes. Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion. Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs. The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come.

The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains—a HECT domain or a RING finger—and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.

The 3-phosphorylated inositol lipids fulfill roles as second messengers by interacting with the lipid binding domains of a variety of cellular proteins. Such interactions can affect the subcellular localization and aggregation of target proteins, and through allosteric effects, their activity. Generation of 3-phosphoinositides has been documented to influence diverse cellular pathways and hence alter a spectrum of fundamental cellular activities. This review is focused on the 3-phosphoinositide lipids, the synthesis of which is acutely triggered by extracellular stimuli, the enzymes responsible for their synthesis and metabolism, and their cell biological roles. Much knowledge has recently been gained through structural insights into the lipid kinases, their interaction with inhibitors, and the way their 3-phosphoinositide products interact with protein targets. This field is now moving toward a genetic dissection of 3-phosphoinositide action in a variety of model organisms. Such approaches will reveal the true role of the 3-phosphoinositides at the organismal level in health and disease.

Recent years have witnessed dramatic advances in our understanding of how newly translated proteins fold in the cell and the contribution of molecular chaperones to this process. Folding in the cell must be achieved in a highly crowded macromolecular environment, in which release of nonnative polypeptides into the cytosolic solution might lead to formation of potentially toxic aggregates. Here I review the cellular mechanisms that ensure efficient folding of newly translated proteins in vivo. De novo protein folding appears to occur in a protected environment created by a highly processive chaperone machinery that is directly coupled to translation. Genetic and biochemical analysis shows that several distinct chaperone systems, including Hsp70 and the cylindrical chaperonins, assist the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells. The cellular chaperone machinery is specifically recruited to bind to ribosomes and protects nascent chains and folding intermediates from nonproductive interactions. In addition, initiation of folding during translation appears to be important for efficient folding of multidomain proteins.

Actin filament assembly and turnover drive many forms of cellular motility, particularly extension of the leading edge of locomoting cells and rocketing of pathogenic microorganisms through host cell cytoplasm. De novo nucleation of actin filaments appears to be required for these movements. A complex of seven proteins called Arp2/3 complex is the best characterized cellular initiator of actin filament nucleation. Arp2/3 complex is intrinsically inactive, relying on nucleation promoting factors for activation. WASp/Scar family proteins are prominent cellular nucleation promoting factors. They bring together an actin monomer and Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction. WASp and N-WASP are intrinsically autoinhibited, and their activity is regulated by Rho-family GTPases such as Cdc42, membrane polyphosphoinositides, WIP/verprolin, and SH3 domain proteins. These interactions provide a final common pathway for many signaling inputs to regulate actin polymerization. Microorganisms either activate Arp2/3 complex directly or usurp N-WASP to initiate actin polymerization.

Genetic, biochemical, and spectroscopic studies have established a new function for an intracellular protein, i.e., guiding and inserting a copper cofactor into the active site of a target enzyme. Studies of these new proteins have revealed a fundamental aspect of copper physiology, namely the vast overcapacity of the cytoplasm for copper sequestration. This finding framed the mechanistic, energetic, and structural aspects of intracellular copper trafficking proteins. One hallmark of the copper chaperones is the similarity of the protein fold between the chaperone and its target enzyme. The surface residues presented by each partner, however, are quite different, and some initial findings concerning the complementarity of these interfaces have led to mechanistic insights. The copper chaperones appear to lower the activation barrier for metal transfer into specific protein-binding sites. The manner in which they facilitate metal insertion appears to involve a docking of the metal donor and acceptor sites in close proximity to one another. Although the intimate mechanism is still open, it appears that a low activation barrier for metal transfer is achieved by a network of coordinate-covalent, electrostatic, and hydrogen bonding interactions in the vicinity of the metal-binding site itself.