9601096 Robertus Histidine decarboxylase (HDC) from Lactobacillus 30a converts histidine to histamine and CO2. The enzyme shows cooperative regulation. It is activated by low pH. The goal is to examine the mechanism of this activation in atomic detail using X-ray crystallography and site-specific mutagenesis. A kinetic analysis of HDC found the wild-type enzyme can be described as having a Tense state T with low substrate affinity and a Relaxed state R with higher affinity; low pH and high histidine concentrations stabilize the R form. Several site-directed mutants, including some double mutants, have been made which stabilize the T form. The X-ray structure of HDC originally solved by the Hackert group corresponds to the R state at pH 4.8. Two mutants of the enzyme have been crystallized in its T state. X-ray data have been collected to 3.1 ( resolution on one form and molecular replacement and SIR phased solutions of the structures are underway. It is also proposed to complete the molecular structure of several other important mutants which have been crystallized. The T structures will be compare with the known R model to describe tertiary and quaternary structural changes involved in the cooperative mechanism. Residues likely to play prominent roles in the T to R conversion will be identified. A substrate analog, histidine methyl ester HME, will be soaked into the crystals and the mode of substrate binding analyzed. These will be compared with the binding already described for R state HDC at pH 4.8. Difference in binding caused by pH and/or by quaternary structural rearrangement will be elucidated. %%% The overall goal of this research is to shed light on the atomic forces and interactions which turn enzymes on and off. The principles involved in this kind of regulation are universal, and what we learn studying an enzyme from bacteria will help us understand the rules used by all living systems. The switch trigger under study in this project, the simple hydrogen ion, is of considerable importance. The reason is that many metabolic processes involve the movement or production of hydrogen ions, and indeed they are among the most common effectors of biologic activity known. In spite of the simplicity and breadth of this kind of enzyme regulation, there are presently no detailed models of how hydrogen ions turn proteins off or on at the atomic level. We have formed a likely hypothesis that the positively charged ion binds between two molecules of the enzyme when they are arranged in a ring, and "pulls" them into an active conformation. When the hydrogen ion level in the cell decreases, the ones trapped in the protein exit leaving two negative charges at this site on the proteins to repel one another. This repulsion changes the enzyme shape into an inactive conformation. The work in this proposal should produce an atomic model of the enzyme with and without the bound hydrogen ion and allow us to actually see if the hypothesis is correct. This in turn will build our confidence that we can understand the strategies used by other living systems to regulate enzyme activity according to the local hydrogen ion concentration (pH). ***
