ABSTRACT
Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
Subject(s)
DNA/chemistry , Histones/chemistry , Nucleosome Assembly Protein 1/chemistry , Nucleosomes/ultrastructure , Animals , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Histones/genetics , Histones/metabolism , Humans , Hydroxyl Radical/chemistry , Models, Molecular , Nucleosome Assembly Protein 1/genetics , Nucleosome Assembly Protein 1/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , Osmolar Concentration , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenopus laevisABSTRACT
Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , DNA/metabolism , Histones/metabolism , Nucleosomes/metabolism , Animals , Base Pairing , Binding Sites , Chromatin/chemistry , Chromatin/genetics , Chromatin/ultrastructure , Cryoelectron Microscopy , DNA/chemistry , DNA/genetics , Histones/chemistry , Humans , Models, Molecular , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/ultrastructure , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Time Factors , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
Self-assembling dipeptides have emerged in the last two decades as promising building blocks for the development of novel biomaterials. Among the various classes of dipeptides, aromatic dipeptides and especially diphenylalanine (Phe-Phe), which forms hexagonal nanotubes, have been the most extensively studied. However, aliphatic peptides or mixed aromatic-aliphatic dipeptides seem just as promising, exhibiting various structures ranging from amyloid fibrils to microtubes. Herein we report the single-crystal structure of an aliphatic dipeptide, alanine-isoleucine (Ala-Ile), C17H24N2O5, protected with a benzyloxycarbonyl (Z) group at the N-terminus. The protected dipeptide crystallizes in the orthorhombic space group P212121 and forms hollow microtubes with orthorhombic symmetry upon evaporation on glass surfaces, as shown by field emission scanning electron microscopy (FESEM). These findings provide an increased understanding of the correlation between the single-crystal structure of the peptide building block and its self-assembly mechanism, and expand the library of available building blocks for microtechnological applications.
Subject(s)
Alanine , Isoleucine , Alanine/chemistry , Hydrogen Bonding , Crystallography, X-Ray , Dipeptides/chemistryABSTRACT
The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.
Subject(s)
Nucleoside-Diphosphate Kinase , Animals , Mice , Nucleoside-Diphosphate Kinase/genetics , Diet, High-Fat/adverse effects , Epigenesis, Genetic , Histones , Liver , Fatty Acids , Mice, KnockoutABSTRACT
The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise is unclear. We exploited HCT116 cancer cell line to select resistant clones to S-trityl-L-cysteine (STLC), an extensively studied Eg5 loop-L5 binding inhibitor. The STLC resistant clones differed in their resistance to other loop-L5 binding inhibitors but remained sensitive to the ATP class of competitive Eg5 specific inhibitors. Eg5 is still necessary for bipolar spindle formation in the resistant clones since the cells were sensitive to RNAi mediated depletion of Eg5. One clone expressing Eg5(T107N), a dominant point mutation in the P-loop of the ATP binding domain of the motor, appeared to be not only resistant but also dependent on the presence of STLC. Eg5(T107N) expression was associated also with resistance to the clinical relevant loop-L5 Eg5 inhibitors, Arry-520 and ispinesib. Ectopic expression of the Eg5(T107N) mutant in the absence of STLC was associated with strong non-exchangeable binding to microtubules causing them to bundle. Biochemical assays showed that in contrast to the wild type Eg5-STLC complex, the ATP binding site of the Eg5(T107N) is accessible for nucleotide exchange only when the inhibitor is present. We predict that resistance can be overcome by inhibitors that bind to other than the Eg5 loop-L5 binding site having different chemical scaffolds, and that allostery-dependent resistance to Eg5 inhibitors may also occur in cells and may have positive implications in chemotherapy since once diagnosed may be beneficial following cessation of the chemotherapeutic regimen.
ABSTRACT
HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.
Subject(s)
HIV-1 , beta Karyopherins , HIV-1/metabolism , Models, Structural , Molecular Docking Simulation , RNA, Viral/metabolism , beta Karyopherins/genetics , beta Karyopherins/metabolismABSTRACT
Eg5, the product of Kif11 gene, also known as kinesin spindle protein, is a motor protein involved in the proper establishment of a bipolar mitotic spindle. Eg5 is one of the 45 different kinesins coded in the human genome of the kinesin motor protein superfamily. Over the last three decades Eg5 has attracted great interest as a promising new mitotic target. The identification of monastrol as specific inhibitor of the ATPase activity of the motor domain of Eg5 inhibiting the Eg5 microtubule motility in vitro and in cellulo sparked an intense interest in academia and industry to pursue the identification of novel small molecules that target Eg5 in order to be used in cancer chemotherapy based on the anti-mitotic strategy. Several Eg5 inhibitors entered clinical trials. Currently the field is faced with the problem that most of the inhibitors tested exhibited only limited efficacy. However, one Eg5 inhibitor, Arry-520 (clinical name filanesib), has demonstrated clinical efficacy in patients with multiple myeloma and is scheduled to enter phase III clinical trials. At the same time, new trends in Eg5 inhibitor research are emerging, including an increased interest in novel inhibitor binding sites and a focus on drug synergy with established antitumor agents to improve chemotherapeutic efficacy. This review presents an updated view of the structure and function of Eg5-inhibitor complexes, traces the possible development of resistance to Eg5 inhibitors and their potential therapeutic applications, and surveys the current challenges and future directions of this active field in drug discovery.
Subject(s)
Antimitotic Agents/pharmacology , Antineoplastic Agents/pharmacology , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Adenosine Triphosphate/metabolism , Animals , Antimitotic Agents/chemistry , Antimitotic Agents/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , Binding Sites , Biological Products/chemistry , Biological Products/pharmacology , Clinical Trials as Topic , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/physiology , Humans , Kinesins/chemistry , Molecular Targeted Therapy/methodsABSTRACT
Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Histone Chaperones/metabolism , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Signal Transduction/genetics , Transcription Factors/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Animals , Cell Proliferation/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Gene Knockout Techniques , Genotype , HeLa Cells , Hep G2 Cells , Humans , Mice , Microorganisms, Genetically-Modified , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , TransfectionABSTRACT
Structures of homologous proteins are usually conserved during evolution, as are critical active site residues. This is the case for actin and tubulin, the two most important cytoskeleton proteins in eukaryotes. Actins and their related proteins (Arps) constitute a large superfamily whereas the tubulin family has fewer members. Unaligned sequences of these two protein families were analysed by searching for short groups of family-specific amino acid residues, that we call motifs, and by counting the number of residues from one motif to the next. For each sequence, the set of motif-to-motif residue counts forms a subfamily-specific pattern (landmark pattern) allowing actin and tubulin superfamily members to be identified and sorted into subfamilies. The differences between patterns of individual subfamilies are due to inserts and deletions (indels). Inserts appear to have arisen at an early stage in eukaryote evolution as suggested by the small but consistent kingdom-dependent differences found within many Arp subfamilies and in gamma-tubulins. Inserts tend to be in surface loops where they can influence subfamily-specific function without disturbing the core structure of the protein. The relatively few indels found for tubulins have similar positions to established results, whereas we find many previously unreported indel positions and lengths for the metazoan Arps.
Subject(s)
Actins/chemistry , Amino Acid Motifs , Tubulin/chemistry , Actins/genetics , Amino Acid Motifs/genetics , Animals , Databases, Protein , Humans , INDEL Mutation , Introns , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Models, Molecular , Tubulin/geneticsABSTRACT
The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.
Subject(s)
CDC2 Protein Kinase/metabolism , Microtubules/metabolism , Mitosis/physiology , Tubulin/metabolism , Amino Acid Sequence , Animals , Antibodies, Phospho-Specific/metabolism , Cattle , HCT116 Cells , HeLa Cells , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Phosphopeptides/metabolism , Phosphorylation , Protein Transport , Recombinant Fusion Proteins/metabolism , Sequence Analysis, Protein , Serine/metabolism , Tubulin/chemistry , Tumor Cells, CulturedABSTRACT
The human kinetochore is a highly complex macromolecular structure that connects chromosomes to spindle microtubules (MTs) in order to facilitate accurate chromosome segregation. Centromere-associated protein E (CENP-E), a member of the kinesin superfamily, is an essential component of the kinetochore, since it is required to stabilize the attachment of chromosomes to spindle MTs, to develop tension across aligned chromosomes, to stabilize spindle poles and to satisfy the mitotic checkpoint. Here we report the 2.5A resolution crystal structure of the motor domain and linker region of human CENP-E with MgADP bound in the active site. This structure displays subtle but important differences compared to the structures of human Eg5 and conventional kinesin. Our structure reveals that the CENP-E linker region is in a "docked" position identical to that in the human plus-end directed conventional kinesin. CENP-E has many advantages as a potential anti-mitotic drug target and this crystal structure of human CENP-E will provide a starting point for high throughput virtual screening of potential inhibitors.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Kinetochores/chemistry , Adenosine Diphosphate/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Determining the mechanism of action of drugs and their target specificity in cells remains a major challenge. Here we describe the use of cell lines expressing two point mutations in the allosteric inhibitor binding pocket of the mitotic kinesin Eg5 (D130A, in the loop L5 region and L214A in helix α3), which following transfection, were selected for their ability to proliferate normally in the presence of STLC, a well known Eg5 inhibitor. The cell lines were used to discriminate the mechanism of action of other chemically distinct small molecule inhibitors of Eg5 that differ in their mode of action. The STLC resistant cells were capable of continuous proliferation in the presence of ATP uncompetitive inhibitors, such as K858 and dimethylenastron, but were still sensitive to ATP competitive inhibitors that are thought to bind to a distinct site on Eg5 than the allosteric binding pocket. The STLC resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs from drugs binding to other pockets without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they have additional targets in dividing cells.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Line, Tumor/drug effects , Cysteine/analogs & derivatives , Drug Resistance, Neoplasm , Kinesins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Allosteric Site , Binding Sites , Cell Line, Tumor/metabolism , Cell Proliferation/drug effects , Cysteine/pharmacology , Humans , Kinesins/antagonists & inhibitors , Kinesins/genetics , M Phase Cell Cycle Checkpoints/drug effects , Point Mutation , Quinazolines/pharmacology , Thiadiazoles/pharmacology , Thiones/pharmacologyABSTRACT
Drugs that target mitotic spindle proteins have been proven useful for tackling tumor growth. Eg5, a kinesin-5 family member, represents a potential target, since its inhibition leads to prolonged mitotic arrest through the activation of the mitotic checkpoint and apoptotic cell death. Monastrol, a specific dihydropyrimidine inhibitor of Eg5, shows stereo-specificity, since predominantly the (S)-, but not the (R)-, enantiomer has been shown to be the biologically active compound in vitro and in cell-based assays. Here, we solved the crystal structure (2.7A) of the complex between human Eg5 and a new keto derivative of monastrol (named mon-97), a potent antimitotic inhibitor. Surprisingly, we identified the (R)-enantiomer bound in the active site, and not, as for monastrol, the (S)-enantiomer. The absolute configuration of this more active (R)-enantiomer has been unambiguously determined via chemical correlation and x-ray analysis. Unexpectedly, both the R- and the S-forms inhibit Eg5 ATPase activity with IC(50) values of 110 and 520 nM (basal assays) and 150 nm and 650 nm (microtubule-stimulated assays), respectively. However, the difference was large enough for the protein to select the (R)- over the (S)-enantiomer. Taken together, these results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors. This considerably broadens the alternatives for rational drug design.
Subject(s)
Antimitotic Agents/pharmacology , Kinesins/antagonists & inhibitors , Kinesins/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Thiones/chemistry , Thiones/pharmacology , Antimitotic Agents/chemistry , Crystallography, X-Ray , Humans , Protein Conformation , StereoisomerismABSTRACT
The Rab small G-protein family plays important roles in eukaryotes as regulators of vesicle traffic. In Rab proteins, the hydrolysis of GTP to GDP is coupled with association with and dissociation from membranes. Conformational changes related to their different nucleotide states determine their effector specificity. The crystal structure of human neuronal Rab6B was solved in its 'inactive' (with bound MgGDP) and 'active' (MgGTPgammaS-bound) forms to 2.3 and 1.8 A, respectively. Both crystallized in space group P2(1)2(1)2(1), with similar unit-cell parameters, allowing the comparison of both structures without packing artifacts. Conformational changes between the inactive GDP and active GTP-like state are observed mainly in the switch I and switch II regions, confirming their role as a molecular switch. Compared with other Rab proteins, additional changes are observed in the Rab6 subfamily-specific RabSF3 region that might contribute to the specificity of Rab6 for its different effector proteins.
Subject(s)
Guanylyl Imidodiphosphate/metabolism , Neurons/enzymology , rab GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Catalysis , Crystallography, X-Ray/methods , Enzyme Activation , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Neurons/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Human centromere-associated protein E, a member of the kinesin superfamily, is a microtubule-dependent motor protein involved in cell division that has been localized transiently to the kinetochore. The protein is thought to be responsible for the correct attachment and positioning of chromosomes to the mitotic spindle during the metaphase. The 312 kDa protein comprises four different domains. In this study, the focus was on the N-terminal motor domain, which includes the ATP-binding site and a region for microtubule binding. Crystals of the CENP-E motor domain have been obtained by high-throughput crystallization screening using an automated TECAN crystallization robot. The crystals (737 x 132 x 79 microm) belong to the space group P2(1), with unit-cell parameters a = 49.35, b = 83.70, c = 94.16 angstroms, beta = 103.05 degrees. They diffract to 2.1 angstroms resolution using synchrotron radiation.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Crystallography, X-Ray/methods , Adenosine Triphosphate/chemistry , Anisotropy , Binding Sites , Chromosomes/ultrastructure , Crystallization , DNA/chemistry , Humans , Mitosis , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Spindle Apparatus/metabolismABSTRACT
The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, d-captopril, has been solved at 1.5-A resolution. The enzyme has the typical alphabeta/betaalpha metallo-beta-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn2+ ions were identified. d-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn2+ ions. Interestingly, d-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn2+ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors.
Subject(s)
Captopril/chemistry , Flavobacterium/chemistry , beta-Lactamases/chemistry , Bacterial Proteins/chemistry , Binding Sites , Captopril/metabolism , Crystallization , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Molecular Structure , Protein Binding , Protein Conformation , Zinc/chemistry , beta-Lactamases/metabolismABSTRACT
The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.