Molecular modeling and site-directed mutagenesis define the catalytic motif in human gamma -glutamyl hydrolase.

Human gamma-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-gamma-glutamate metabolism, which functions by catalyzing the cleavage of the gamma-glutamyl chain of substrates. We previously reported that Cys-110 is essential for activity. Using the sequence of hGH as a query, alignment searches of protein data bases were made using the SSearch and TPROBE programs. Significant similarity was found between hGH and the glutamine amidotransferase type I domain of Escherichia coli carbamoyl phosphate synthetase. The resulting hypothesis is that the catalytic fold of hGH is similar to the folding of this domain in carbamoyl phosphate synthetase. This model predicts that Cys-110 of hGH is the active site nucleophile and forms a catalytic triad with residues His-220 and Glu-222. The hGH mutants C110A, H220A, and E222A were prepared. Consistent with the model, mutants C110A and H220A were inactive. However, the V(max) of the E222A hGH mutant was reduced only 6-fold relative to the wild-type enzyme. The model also predicted that His-171 in hGH may be involved in substrate binding. The H171N hGH mutant was found to have a 250-fold reduced V(max). These studies to determine the catalytic mechanism begin to define the three dimensional interactions of hGH with poly-gamma-glutamate substrates.

Identification and characterization of two groups of congenital hypothyroid infants: implications for newborn screening.

A retrospective analysis of 400 newborns diagnosed with congenital primary hypothyroidism between 1983 and 1987 was conducted. Two distinct groups of cases were identified and characterized based on their newborn screening TSH value. The two groups are separated at a TSH concentration of 50 mU/l of serum by a normal probability plot. This finding is in agreement with the 1993 recommendation from the American Academy of Pediatrics that infants with a low T4 level and a TSH concentration greater than 40 mU/l be considered to have primary hypothyroidism until proven otherwise. The group of infants with TSH less than 50 mU/l have a higher proportion of males and low birthweight infants. For this group, T4 increases with increasing TSH. We find that screening TSH, T4, and birthweight are predictive of follow-up serum TSH level for the cases with TSH > 50 mU/l but not for cases with TSH < 50 mU/l. An optimal rule for selecting screening cutoff levels is presented based on only T4, TSH and their interaction. Adjustments for sex, birthweight or age at which sample was taken did not aid in distinguishing cases from controls for newborns whose age of sample is 2 days or greater.

Identification of the most significant amphipathic helix with application to HIV and MHV envelope proteins.

Amphipathic helices, which play important roles in protein structure, occur in a wide variety of lengths. Yet existing methods employ fixed window lengths. We present a hierarchical procedure that identifies the Q most significant amphipathic helices regardless of length. Since the observed hydrophobicities are not normally distributed, test statistics usually employed for least-squares regression are inappropriate for assessing statistical significance of amphipathic helices. We show that an adjusted F statistic provides a good test. An application to the envelope protein of HIV finds an unexpected long amphipathic helix in gp41.

Algorithms for the optimal identification of segment neighborhoods.

Two algorithms for the efficient identification of segment neighborhoods are presented. A segment neighborhood is a set of contiguous residues that share common features. Two procedures are developed to efficiently find estimates for the parameters of the model that describe these features and for the residues that define the boundaries of each segment neighborhood. The algorithms can accept nearly any model of segment neighborhood, and can be applied with a broad class of best fit functions including least squares and maximum likelihood. The algorithms successively identify the most important features of the sequence. The application of one of these methods to the haemagglutinin protein of influenza virus reveals a possible mechanism for conformational change through the finding of a break in a strong heptad repeat structure.