Enzymology of Phosphorus Metabolism

Gerlt, John A.

Abstract

The importance of adenylate cyclase in the regulation and mediation of a diverse array of biological and biochemical processes requires that the chemical mechanism of the reaction it catalyzes be understood. This information may allow the design and synthesis of novel suicide inhibors of the enzyme which can be used both in vitro and in vivo to better understand the mechanisms by which cyclic nucleotides exert their effects. In order to characterize the mechanism of adenylate cyclase, we are preparing ATP which is oxygen chiral at either the alpha- or beta-phosphorus atom; these materials will be useful in additional stereochemical studies of the reaction and in defining the role of metal ions in catalysis. We also plan to determine whether adenylate cyclase contains tightly bound metal ions which are required for activity, since our preliminary experiments and data available for enzymes which catalyze similar reactions indicate that such metal ions may be absolutely required for catalytic activity.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of General Medical Sciences (NIGMS)
• Type: Research Project (R01)
• Project #: 5R01GM034572-03
• Application #: 3285825
• Study Section: Biochemistry Study Section (BIO)
• Project Start: 1984-08-01
• Project End: 1990-07-31
• Budget Start: 1986-08-01
• Budget End: 1987-07-31
• Support Year: 3
• Fiscal Year: 1986

Institution

• Name: University of Maryland College Park
• Type: Earth Sciences/Resources
• City: College Park
• State: MD
• Country: United States
• Zip Code: 20742

Publications

Landro, J A; Gerlt, J A; Kozarich, J W et al. (1994) The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate. Biochemistry 33:635-43

Gerlt, J A; Gassman, P G (1993) Understanding the rates of certain enzyme-catalyzed reactions: proton abstraction from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters. Biochemistry 32:11943-52

Mitra, B; Gerlt, J A; Babbitt, P C et al. (1993) A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry 32:12959-67

Petsko, G A; Kenyon, G L; Gerlt, J A et al. (1993) On the origin of enzymatic species. Trends Biochem Sci 18:372-6

Mazumder, A; Gerlt, J A; Absalon, M J et al. (1991) Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry 30:1119-26

Powers, V M; Koo, C W; Kenyon, G L et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism. Biochemistry 30:9255-63

Landro, J A; Kallarakal, A T; Ransom, S C et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant. Biochemistry 30:9274-81

Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1990) Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry 29:9856-62

Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1989) Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme. Biochemistry 28:969-75

Holland, M M; Leib, T K; Gerlt, J A (1988) Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin. J Biol Chem 263:14661-8