In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.
The sulfonation of endobiotics and xenobiotics is a fundamental metabolic process of major importance. The sulfoconjugation of biomolecules occurs widely, involves compounds with molecular weights ranging in size from Ͻ10 3 to Ͼ10 6 and results in a dramatic change in the physicochemical property of the sulfonated compounds (1). Sulfonated macromolecules such as glycosaminoglycans and proteoglycans are involved in cell surface structure and connective tissue. The sulfonation of tyrosine residues has been established as a widespread posttranslational modification for many secretory and membrane proteins. Sulfolipids such as sphingolipids and galactoglycerolipids are concentrated in the brain, peripheral nerves, and reproductive tissues. Additionally, sulfoconjugation is important in the biotransformation of many endogenous low molecular weight compounds such as neurotransmitters and hormones, e.g. catecholamines, iodothyronines, and steroids. The sulfonation of drugs and xenobiotics functions primarily to inactivate and clear these generally hydrophobic compounds from the body, although there are examples where the active form of a drug is sulfonated.In the course of sulfonation, inorganic sulfate must be activated prior to being transferred to an acceptor molecule (2). In mammalian metabolism, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) 1 has been identified as the activated sulfate molecule (3) and represents the universal sulfonate (SO 3 Ϫ ) donor for all sulfotransferase reactions (4). The activation of inorganic sulfate to form PAPS results from the concerted action of two enzymes (3,5,6). The first step is catalyzed by ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and involves the reaction of inorganic sulfate with ATP to form adenosine-5Ј-phosphosulfate (APS) and inorganic pyrophosphate (PP i ). This reaction results in the formation of a high energy phosphoricsulfuric acid anhydride bond that is the chemical basis for sulfate activation (8). In the direction of APS formation the enzyme has an unfavorable equilibrium (K eq ϳ 10 Ϫ7 M) (9); it has been suggested that the reaction is driven in the physiologic direction by the hydrolysis of PP i by the action of a ubiquitous inorganic pyrophosphatase (9). The second step is catalyzed by APS kinase (ATP:adenylylsulfate 3Ј-phosphotransferase, EC 2.7.1.25) and involves the reaction of APS with ATP to form PAPS and ADP. Unlike ATP sulfurylase, APS kinase is not involved in the activation of sulfate, and its raison d'être is not known (8).ATP sulfurylase and APS kinase cloned from bacteria (10 -13), fungi (14, 15), yeast (16, 17), and plants (18, 19) are found on separate polypeptide chains. In these species the molecular mass of ATP sulfurylase ranges from 35.2 to 63.9 kDa, whereas the molecular mass of APS kinase ranges from 22.3 to 29.7 kDa. In contrast to what is found in bacteria, fungi, yeast, and plants, however, ATP sulfurylase and APS kinase isolated from mammalian tissues, e.g. rat chondrosarcoma (20) and guinea pig adrenal 2 are physica...
Summary3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase (PAPSS) catalyzes the biosynthesis of PAPS which serves as the universal sulfonate donor compound for all sulfotransferase reactions. PAPSS forms PAPS in two sequential steps. First inorganic sulfate combines with ATP to form adenosine 5'-phosphosulfate (APS) and pyrophosphate catalyzed by ATP sulfurylase domain and in the second step, APS combines with another molecule of ATP to form PAPS and ADP catalyzed by APS kinase domain. The bifunctional PAPSS1 is comprised of NH 2 -terminal APS kinase domain (*1-260 aa), and a COOH-terminal ATP sulfurylase domain (*220-623 aa). In humans there are two major isoforms PAPSS1 and PAPSS2. In brain and skin PAPSS1 is the major expressed isoform, whereas in liver, cartilage and adrenal glands PAPSS2 isoform expression predominates and in various other tissues the proportions of the isoform expressions is purported to vary. The deduced amino acid sequences of the two isoforms reveal 77% identity between PAPSS1 and PAPSS2. In addition there is a splice variant PAPSS2b which contains notably an extra five amino acid sequence GMALP. From human tissues PAPSS1 and a splice variant PAPSS2b has been molecularly cloned, overexpressed, purified and have been biochemically characterized partially. PAPSS2b exhibited an apparent difference towards varying ATP concentration showing a sigmoidal response, with a 0.5 [v/V max ] at 1.4 mM ATP whereas PAPSS1 exhibited a hyperbolic response with a 0.5 [v/V max ] at 0.25 mM ATP. Although this being the case, comparison of PAPSS1 and PAPSS2 crude extracts, did not show marked difference in the kinetic properties with either substrates ATP or sulfate leading to speculate that the extra GMALP pentapeptide present in PAPSS2b could be altering the kinetic behavior. The ATP binding sites of the a-b-ATP hydrolysis, active site motif HxxH (425-428 aa) is present in the ATP sulfurylase domain and the b-g-hydrolase motif GxxGxxK (59-65 aa) is present in the APS kinase domain. The motifs are highly conserved between both isoforms. Gene sequence analysis of PAPSS1 (*106 kB) and PAPSS2 (*86.5 kB), revealed a total of 12 exons. Among exons 2-11 the sizes are highly conserved, although intron sizes varied remarkably. Exons 1 and 12 varied in sizes, contained 5'-UTR and 3'-UTR respectively. PAPSS1 and PAPSS2 contained no putative TATA box and CCAAT box. However both PAPSS1 and PAPSS2 possessed many GC boxes. From promoter analysis, it is apparent that both PAPSS1 and PAPSS2 are inducible, perhaps at various time periods, regulated by specific transcription factors. The deficiency of PAPSS2 results in osteochondrodysplasias. Osteochondrodysplasias are genetically heterogeneous group of disorders that affects skeletal development, linear growth, and the maintenance of cartilage and bone. A large inbred family with a distinct form of recessively inherited, spondyloepimetaphyseal dysplasia (SEMD) was mapped to PAPSS2 isoform located in the chromosome region of 10q23-24. PAPSS1 located in the chromosome 4q2...
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