Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity.

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.

Chain elongation in the isoprenoid biosynthetic pathway.

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.

Regulation of product chain length by isoprenyl diphosphate synthases.

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C15), geranylgeranyl (C20), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C25) diphosphate (F113S), and longer chain prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure of the F112A/F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 A as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117-D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.

Tennis elbow: a review.

Tennis elbow is a common yet sometimes complex musculoskeletal condition affecting many patients treated by physical therapists. The purpose of this article is to review the anatomy, clinical examination, differential diagnosis, conservative care, and surgical treatment for tennis elbow or lateral epicondylitis. Particular attention is given to determining the precise pathological cause of lateral epicondylitis, with consideration of intrinsic and extrinsic factors associated with this condition. This information should assist health care practitioners who treat patients with this disorder.

Rh sensitization after genetic amniocentesis.

A retrospective evaluation was undertaken of 78 Rh-negative women who underwent genetic amniocentesis without Rh-immune globulin prophylaxis. Of the 56 patients at risk for sensitization, 3 (5.4%) became sensitized during the pregnancy in which amniocentesis was performed. This number is not statistically different from the 2.1% incidence of spontaneous Rh immunization during pregnancy. However, a trend toward increasing sensitization after second-trimester amniocentesis was noted. As the fetoplacental blood volume at 16 weeks' gestation is approximately 12 to 13 ml, a 150-micrograms dose of Rh-immune globulin is recommended for the Rh-negative patient who is undergoing genetic amniocentesis.

Prenatal diagnosis by amniocentesis in 800 pregnancies.

Prenatal diagnoses by amniocentesis have been made in 800 pregnancies at the Genetics Division of the Los Angeles County-University of Southern California Medical Center. This experience has indicated that the amniocentesis procedure is safe and highly accurate. Amniocentesis was not associated with significant morbidity or mortality for either infant or mother. The observed complications are assumed to be related to the high-risk nature of these pregnancies, the patients having been selected primarily on the basis of advanced maternal age or a previous abnormal child. The needle puncture marks, which occurred in 2.4 percent of the live births, resulted in no serious developmental or cosmetic effects to the infants. No errors in cytogenetic diagnosis are known to have occurred in this series.

Needle puncture of fetus: a complication of second-trimester amniocentesis.

Needle puncture of the fetus has rarely been reported with midtrimester amniocentesis. This paper contains the report of five cases of needle scars in infants born after second-trimester amniocentesis for prenatal diagnosis of fetal genetic disorders. Since this complication may be more frequent than has been previously believed, there is the possibility that damage to the fetus may occur. It is suggested that the products of all abortions and all live-born and stillborn infants delivered following amniocentesis should be examined for evidence of injury.